Nucleic acids encoding D-type cyclins and hybridization probes

ABSTRACT

The present invention relates to a novel class of cyclins, referred to as D-type cyclins, of mammalian origin, particularly human origin, DNA and RNA encoding the novel cyclins, and a method of identifying other D-type and non-D type cyclins. Also disclosed are a method of detecting an increased level of a D-type cyclin and a method of inhibiting cell division by interfering with formation of the protein kinase-D type cyclin complex essential for cell cycle start.

FUNDING

Work described herein was supported by National Institutes of Health Grant GM39620 and the Howard Hughes Medical Institute. The United States Government has certain rights in the invention.

RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 07/963,308 filed on Oct. 16, 1992, which is a continuation-in-part of U.S. Ser. No. 07/888,178 filed May 26, 1992 now abandoned and entitled "D-Type Cyclin and Uses Related Thereto", which corresponds to and claims priority to Patent Cooperation Treaty Application No. PCT/US92/04146, filed May 18, 1992 and entitled "D-Type Cyclin and Uses Related Thereto", and which is a continuation-in-part of U.S. Ser. No. 07/701,514, filed May 16, 1991 and entitled "D-Type Cyclin and Uses Related Thereto." The teachings of U.S. Ser. Nos. 07/888,178, 07/701,514 and the PCT Application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A typical cell cycle of a eukaryotic cell includes the M phase, which includes nuclear division (mitosis) and cytoplasmic division or cytokinesis and interphase, which begins with the G1 phase, proceeds into the S phase and ends with the G2 phase, which continues until mitosis begins, initiating the next M phase. In the S phase, DNA replication and histone synthesis occurs, while in the G1 and G2 phases, no net DNA synthesis occurs, although damaged DNA can be repaired. There are several key changes which occur during the cell cycle, including a critical point in the G1 phase called the restriction point or start, beyond which a cell is committed to completing the S, G2 and M phases.

Onset of the M phase appears to be regulated by a common mechanism in all eukaryotic cells. A key element of this mechanism is the protein kinase p34^(cdc2), whose activation requires changes in phosphorylation and interaction with proteins referred to as cyclins, which also have an ongoing role in the M phase after activation.

Cyclins are proteins that were discovered due to their intense synthesis following the fertilization of marine invertebrate eggs (Rosenthal, E. T. et al., Cell 20:487-494 (1980)). It was subsequently observed that the abundance of two types of cyclin, A and B, oscillated during the early cleavage divisions due to abrupt proteolytic degradation of the polypeptides at mitosis and thus, they derived their name (Evans, T. et al., Cell 33:389-396 (1983); Swenson, K. I. et al., Cell 47:867-870 (1986); Standart, N. et al., Dev. Biol. 124:248-258 (1987)).

Active rather than passive involvement of cyclins in regulation of cell division became apparent with the observation that a clam cyclin mRNA could cause activation of frog oocytes and entry of these cells into M phase (Swenson, K. I. et al., Cell 7:867-870 (1986)). Activation of frog oocytes is associated with elaboration of an M phase inducing factor known as MPF (Masui, Y. and C. L. Markert, J. Exp. Zool. 177:129-146 (1971); Smith, L. D. and R. E. Ecker, Dev. Biol. 25:232-247 (1971)). MPF is a protein kinase in which the catalytic subunit is the frog homolog of the cdc2 protein kinase (Dunphy, W. G. et al., Cell 54:423-431 (1988); Gautier, J. et al., Cell 54:433-439 (1988); Arion, D. et al., Cell 55:371-378 (1988)).

Three types of classes of cyclins have been identified to date: B, A and CLN cyclins. The B-type cyclin has been shown to act in mitosis by serving as an integral subunit of the cdc2 protein kinase (Booher, R. and D. Beach, EMBO J. 6:3441-3447 (1987); Draetta, G. et al., Cell 56:829-838 (1989); Labbe, J. C. et al., Cell 57:253-263 (1989); Labbe, J. C. et al., EMBO J. 8:3053-3058 (1989); Meier, L. et al., EMBO J. 8:2275-2282 (1989); Gautier, J. et al., Cell 60:487-494 (1990)). The A-type cyclin also independently associates with the cdc2 kinase, forming an enzyme that appears to act earlier in the division cycle than mitosis (Draetta, G. et al., Cell 56:829-838 (1989); Minshull, J. et al., EMBO J. 9:2865-2875 (1990); Giordano, A. et al., Cell 58:981-990 (1989); Pines, J. and T. Hunter, Nature 346:760-763 (1990)). The functional difference between these two classes of cyclins is not yet fully understood.

Cellular and molecular studies of cyclins in invertebrate and vertebrate embryos have been accompanied by genetic studies, particularly in ascomycete yeasts. In the fission yeast, the cdc13 gene encodes a B-type cyclin that acts in cooperation with cdc2 to regulate entry into mitosis (Booher, R. and D. Beach, EMBO J., 6:3441-3447 (1987); Booher, R. and D. Beach, EMBO J. 7:2321-2327 (1988); Hagan, I. et al., J. Cell Sci. 91:587-595 (1988); Solomon, M., Cell 54:738-740 (1988); Goebl, M. and B. Byers, Cell 54:433-439 (1988); Booher, R. N. et al., Cell 58:485-497 (1989)).

Genetic studies in both the budding yeast and fission yeast have revealed that cdc2 (or CDC28 in budding yeast) acts at two independent points in the cell cycle: mitosis and the so-called cell cycle "start" (Hartwell, L. H., J. Mol. Biol., 104:803-817 (1971); Nurse, P. and Y. Bissett, Nature 292:558-560 (1981); Piggot, J. R. et al., Nature 298:391-393 (1982); Reed, S. I. and C. Wittenberg, Proc. Nat. Acad. Sci. USA 87:5697-5701 (1990)).

In budding yeast, the start function of the CDC28 protein also requires association of the catalytic subunit of the protein kinase with ancillary proteins that are structurally related to A and B-type cyclins. This third class of cyclin has been called the C1n class, and three genes comprising a partially redundant gene family have been described (Nash, R. et al., EMBO J. 7:4335-4346 (1988); Hadwiger, J. A. et al., Proc. Natl. Acad. Sci. USA 86:6255-6259 (1989); Richardson, H. E. et al., Cell 59:1127-1133 (1989)). The CLN genes are essential for execution of start and in their absence, cells become arrested in the G1 phase of the cell cycle. The CLN1 and CLN2 transcripts oscillate in abundance through the cell cycle, but the CLN3 transcript does not. In addition, the CLN2 protein has been shown to oscillate in parallel with its mRNA (Nash, R. et al., EMBO J. 7:4335-4346 (1988); Cross, F. R., Mol. Cell. Biol. 8:4675-4684 (1988); Richardson, H. E. et al., Cell 59:1127-1133 (1988); Wittenberg, et al., 1990)).

Although the precise biochemical properties conferred on cdc2/CDC28 by association with different cyclins have not been fully elaborated, genetic studies of cyclin mutants clearly establishes that they confer "G1" and "G2" properties on the catalytic subunit (Booher, R. and D. Beach, EMBO J. 6:3441-3447 (1987); Nash, R. et al., EMBO J. 7:4335-4346 (1988); Richardson, H. E. et al., Cell 56:1127-1133 (1989)).

cdc2 and cyclins have been found not only in embryos and yeasts, but also in somatic human cells. The function of the cdc2/cyclin B enzyme appears to be the same in human cells as in other cell types (Riabowol, K. et al., Cell 57:393-401 (1989)). A human A type cyclin has also been found in association with cdc2. No CLN type cyclin has yet been described in mammalian cells. A better understanding of the elements involved in cell cycle regulation and of their interactions would contribute to a better understanding of cell replication and perhaps even alter or control the process.

SUMMARY OF THE INVENTION

The present invention relates to a novel class of cyclins, referred to as D-type cyclins, which are of mammalian origin and are a new family of cyclins related to, but distinct from, previously described A, B or CLN type cyclins. In particular, it relates to human cyclins, encoded by genes shown to be able to replace a CLN-type gene essential for cell cycle start in yeast, which complement a deficiency of a protein essential for cell cycle start and which, on the basis of protein structure, are on a different branch of the evolutionary tree from A, B or CLN type cyclins. Three members of the new family of D-type cyclins, referred to as the human D-type cyclin gene family, are described herein. They encode small (33-34 KDa) proteins which share an average of 57% identity over the entire coding region and 78% in the cyclin box. One member of this new cyclin family, cyclin D1 or CCND1, is 295 amino acid residues and has an estimated molecular weight of 33,670 daltons (Da). A second member, cyclin D2 or CCND2, is 289 amino acid residues and has an estimated molecular weight of 33,045 daltons. It has been mapped to chromosome 12p band p13. A third member, cyclin D3 or CCND3, is 292 amino acid residues and has an estimated molecular weight of approximately 32,482 daltons. It has been mapped to chromosome 6p band p21. The D-type cyclins described herein are the smallest cyclin proteins identified to date. All three cyclin genes described herein are interrupted by an intron at the same position. D-type cyclins of the present invention can be produced using recombinant techniques, can be synthesized chemically or can be isolated or purified from sources in which they occur naturally. Thus, the present invention includes recombinant D-type cyclins, isolated or purified D-type cyclins and synthetic D-type cyclins. Two of the three novel D-type cyclins (cyclin D1 and cyclin D3) have been shown to bind to a novel cyclin dependent kinase (CDK), designated CDK5, which is also the subject of the present invention. Using the methods described herein and an appropriate test system, such as a cell line which expresses cyclin D2, it is possible to determine whether cyclin D2 also binds CDK5. Unlike other cyclin dependent kinases, CDK5 has a PSSALRE motif (amino acid sequence 45-51), rather than the PSTAIRE motif which is conserved in other known members of the CDK family. CDK5 has been shown to be expressed in all cultured cells examined thus far and, therefore, it seems likely that it may perform important, yet unique, role(s) in the cell cycle.

The present invention also relates to DNA or RNA encoding a D-type cyclin of mammalian origin, particularly of human origin, as well as to antibodies, both polyclonal and monoclonal, specific for a D-type cyclin of mammalian, particularly human, origin. Antibodies specific for each of the D-type cyclins described specifically herein (cyclin D1, cyclin D2 and cyclin D3) are in particular the subject of the present invention.

The present invention further relates to a method of isolating genes encoding other cyclins, such as other D-type cyclins and related (but non-D type) cyclins. It also has diagnostic and therapeutic aspects. For example, it relates to a method in which the presence and/or quantity of a D-type cyclin (or cyclins) in tissues or biological samples, such as blood, urine, feces, mucous or saliva, is determined, using a nucleic acid probe based on a D-type cyclin gene or genes described herein or an antibody specific for a D-type cyclin. This embodiment can be used to predict whether cells are likely to undergo cell division at an abnormally high rate (i.e., if cells are likely to be cancerous), by determining whether their cyclin levels or activity are elevated (elevated level of activity being indicative of an increased probability that cells will undergo an abnormally high rate of division). The present method also relates to a diagnostic method in which the occurrence of cell division at an abnormally high rate is assessed based on abnormally high levels of a D-type cyclin(s), a gene(s) encoding a D-type cyclin(s) or a transcription product(s) (RNA).

In addition, the present invention relates to a method of modulating (decreasing or enhancing) cell division by altering the activity of at least one D-type cyclin, such as D1, D2 or D3, the activity of another molecule or molecules with which D-type cyclin associates or interacts, or the activity of both in cells. The present invention particularly relates to a method of inhibiting increased cell division by interfering with the activity or function of a D-type cyclin(s) or of a molecule(s) with which a D-type cyclin associates or interacts. D-type cyclins of the present invention have been shown to associate, in eukaryotic cells, particularly human cells, with multiple cyclin dependent kinases. They have also been shown to co-precipitate with three polypeptides: a cyclin-dependent kinase, a well characterized DNA replication and repair factor (i.e., proliferating cell nuclear antigen or PCNA) and a polypeptide of 21 kDa apparent molecular weight. Results suggest that D-type cyclin, CDK, PCNA and p21 exist in a quaternary complex, that many combinatorial variations of the components 30 (e.g., cyclin D1 or D3 and CDK2, CDK4 and CDK5) assemble in vivo and that each of the quaternary complexes may have a subtly different role in the cell cycle or in different cell types. This knowledge serves as the basis for a variety of approaches to modulating cell division by altering the activity (directly or indirectly) of a D-type cyclin. In one embodiment, it offers specificity in modulating cell division (i.e., the ability to selectively alter cell division in particular cell types or at a particular point in the cycle) because of the specificity of expression of D-type cyclins in cells and the number of possible combinations of the components of the quaternary complex which appear to be formed by D-type cyclin, CDK, PCNA and p21. In a second embodiment, it offers a means by which cell division can be non-specifically altered by interfering with a common component of the quaternary complex of which D-type cyclin is a constituent, such as by interfering with PCNA.

For example, in one embodiment of a therapeutic method of the present invention, function of D-type cyclin(s) is blocked (totally or partially) by interfering with its ability to activate the protein kinase it would otherwise (normally) activate (e.g., p34 or a related protein kinase), by means of agents which interfere with D-type cyclin activity, either directly or indirectly. Such agents include anti-sense sequences or other transcription modulators which bind D cyclin-encoding DNA or RNA; antibodies which bind either the D-type cyclin or a molecule with which a D-type cyclin must interact or bind in order to carry out its role in cell cycle start; substances which bind the D-type cyclin(s); agents (e.g., proteases) which degrade or otherwise inactivate the D-type cyclin(s); or agents (e.g., low molecular weight inhibitors, small organic molecules) which interfere with association of the D-type cyclin with the catalytic subunit of the kinase or inactivate the catalytic subunit itself. In another embodiment, formation of the quaternary complex described above is prevented or enhanced or the activity of a complex member is altered as an approach to altering cell division. Here, too, agents which act indirectly or directly to prevent or enhance complex formation or to alter a constituent's activity can be used. For example, as described above, catalytic activity can be inhibited by preventing activation of the protein kinase. Alternatively, PCNA inhibitors can be introduced into cells in which cell cycle start is to be inhibited, resulting in inhibition of cell division. PCNA inhibitors can act indirectly (e.g., to reduce production of PCNA by interfering with transcription or translation) or directly (e.g., to bind PCNA and prevent it from joining with other complex members). Inhibitors of p21 can also be introduced into cells and interfere, indirectly or directly, with p21 function and/or binding to the complex members. Protein-protein interactions (between or among complex components) can also be altered (reduced or enhanced) to have the desired effect on the cell cycle (to reduce or increase cell division). Agents which block such protein-protein interactions can be used. These include low molecular weight inhibitors, agents which bind to complex components (e.g., antibodies) and agents which degrade or otherwise destroy a component's ability to form a complex with the other proteins. If enhanced quaternary complex formation is desired, agents which increase the ability of complex members to interact and bind (e.g., agents which change the configuration of a complex component so that it is more available for protein-protein interactions necessary for complex formation can be introduced into cells). Enhanced complex formation can also be brought about by increasing in cells the number, activity or availability of the limiting member(s) of the quaternary complex, thus enhancing the rate at which it is formed and its availability to act.

The subject invention also related to agents (e.g., oligonucleotides, antibodies, peptides) useful in the isolation, diagnostic or therapeutic methods described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a genetic screen for human cyclin genes.

FIGS. 2A and 2B show the human cyclin D1 nucleic acid sequence (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2), in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.

FIGS. 3A and 3B show the human cyclin D2 nucleic acid sequence (SEQ ID No. 3) and amino acid sequence (SEQ ID No. 4) in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.

FIGS. 4A and 4B show the human cyclin D3 nucleic acid sequence (SEQ ID No. 5) and amino acid sequence (SEQ ID No. 6), in which nucleotide numbers and amino acid numbers are on the right, amino acid numbers are given with the initiation methionine as number one and the stop codon is indicated by an asterisk.

FIG. 5A shows the amino acid sequence alignment of seven cyclin genes (CYCD1-Hs, SEQ ID No. 7; CYCA-Hs, SEQ ID No. 8; CYCA-Dm, SEQ ID No. 9; CYCB1-Hs, SEQ ID No. 10; CDC13-Sp, SEQ ID No. 11; CLN1-Sc, SEQ ID No. 12; CLN3-Sc, SEQ ID No. 13), in which numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion.

FIG. 5B is a schematic representation of the evolutionary tree of the cyclin family, constructed using the Neighbor-Joining method; the length of horizontal line reflects the divergence.

FIG. 6A is a comparison of several cDNA clones isolated from different cell lines. Open boxes represent the 1.7 kb small transcript containing the coding region of cyclin D1 gene. Shadowed boxes represent the 3' fragment present in the 4.8 kb long transcript. Restriction sites are given above each cDNA clone to indicate the alignment of these clones.

FIG. 6B shows the nucleotide sequence surrounding the first polyadenylation site for several cDNA clones (CYCD1-21, SEQ ID No. 14; CYCD1-H12, SEQ ID No. 15; CYCD1-HO34, SEQ ID No. 16; CYCD1-TO78, SEQ ID No. 17 and a genomic clone; CYCD1-GO68, SEQ ID No. 18).

FIG. 6C is a summary of the structure and alternative polyadenylation of the cyclin D1 gene. Open boxes represent the small transcript, the shadowed box represents the 3' sequence in the large transcript and the filled boxes indicate the coding regions.

FIGS. 7-7D show the protein sequence comparison of eleven mammalian cyclins (CYCD1-Hs, SEQ ID No. 19; CYL1-Mm, SEQ ID No. 20; CYCD2-Hs, SEQ ID No. 21; CYCL2-Mm, SEQ ID No. 22; CYCD3-Hs, SEQ ID No. 23; CYL3-Mm, SEQ ID No. 24; CYCA-Hs, SEQ ID No. 25; CYCB1-Hs, SEQ ID No. 26; CYCB2-Hs, SEQ ID No. 27; CYCC-Hs, SEQ ID No. 28; CYCE-Hs, SEQ ID No. 29).

FIGS. 8A-8C show a schematic representation of the genomic structure of human cyclin D genes, in which each diagram represents one restriction fragment from each cyclin D gene that has been completely sequenced. Solid boxes indicate exon sequences, open boxes indicate intron or 5' and 3' untranslated sequences and hatched boxes represent pseudogenes. The positions of certain restriction sites, ATG and stop codons are indicated at the top of each clone.

FIGS. 9A and 9B show the nucleic acid sequence (SEQ ID No. 30) and amino acid sequence (SEQ ID No. 31, 47, 48 and 49) of a cyclin D2 pseudogene.

FIGS. 10A and 10B show the nucleic acid sequence (SEQ ID Nos. 33 and 50 32) and the amino acid sequence (SEQ ID No. 33) of a cyclin D3 pseudogene.

FIG. 11 is the nucleic acid sequence (SEQ ID No. 34) of 1.3 kb of human cyclin D1 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide-160.

FIG. 12 is the nucleotide sequence (SEQ ID No. 35) of 1.6 kb of human cyclin D2 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide-170.

FIGS. 13A and 13B show the nucleotide sequence (SEQ ID No. 36) of 3.2 kb of human cyclin D3 promoter; the sequence ends at initiation ATG codon and transcription starts at approximately nucleotide-160.

FIGS. 14A and 14B show the nucleotide sequence (SEQ ID No. 37) of human CDK4 cDNA, which encodes an open reading frame of 292 amino acid residues which are shown in single-letter code (SEQ ID No. 37). The underlined peptide (right amino acid residues) at the carboxy-terminus of CDK4 was synthesized to generate peptide antibody.

FIG. 15A: ³⁵ S-methionine-labelled 293 (lanes 1, 2 and 3) or WI38 (lanes 4, 5 and 6) cell lysates were immunoprecipitated with pre-immune serum (lanes 1 and 4), anti-cyclin D1 antiserum (lanes 2 and 5) and anti-CDK2 anti-serum (lanes 3 and 6).

FIG. 15B: ³⁵ S-methionine-labelled WI38 cell lysate was precipitated with pre-immune (lane 1), anti-cyclin D1 (lane 2), and anti-CDK4 antiserum.

FIG. 15C: ³⁵ S-methionine-labelled WI38 cell lysates were immunoprecipitated with pre-immune serum (lane 1), anti-CDK5 peptide antibody in the absence (lane 2) or in the presence of competing CDK5 peptide (lane 3), anti-cyclin D1 (lane 4), or anti-PCNA (lane 5) antisera. The immunoprecipitated polypeptides were analyzed in each case by SDS-polyacrylamide gel electrophoresis as described in Experimental Procedures. The mobility of protein molecular weight standards (BRL) and relevant proteins are indicated.

FIG. 16A is a summary of established pair-wise protein-protein interactions, in which each arrow indicates a demonstrated co-precipitation between two proteins.

FIG. 16B is a schematic representation of the proposed quaternary complex between D-type cyclins, CDKs, p21 and PCNA.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, a new class of mammalian cyclin proteins, designated D-type cyclins, has been identified, isolated and shown to serve as a control element for the cell cycle start, in that they fill the role of a known cyclin protein by activating a protein kinase whose activation is essential for cell cycle start, an event in the G1 phase at which a cell becomes committed to cell division. Specifically, human D-type cyclin proteins, as well as the genes which encode them, have been identified, isolated and shown to be able to replace CLN type cyclin known to be essential for cell cycle start in yeast. The chromosomal locations of CCND2 and CCND3 have also been mapped.

As a result, a new class of cyclins (D type) is available, as are DNA and RNA encoding the novel D-type cyclins, antibodies specific for (which bind to) D-type cyclins and methods of their use in the identification of additional cyclins, the detection of such proteins and oligonucleotides in biological samples, the inhibition of abnormally increased rates of cell division and the identification of inhibitors of cyclins. In addition, two novel D-type cyclins have been shown to bind to a novel cyclin dependent kinase, designated CDK5 and described herein. CDK5 has been shown to differ from all known members of the CDK kinase family in that it has a PSSALRE motif (amino acid residues 45-51) and not the PSTAIRE motif which is conserved among all other known CDKs.

Further, as described herein, Applicant has determined that in eukaryotic cells, specifically human cells, D-type cyclin associates with multiple catalytic subunits (cyclin dependent kinases or CDK). Applicant has also shown that D-type cyclin and CDK co-precipitate with two additional polypeptides: a well characterized DNA replication and repair factor (i.e., proliferating cell nuclear antigen or PCNA) and a polypeptide of 21 kDa apparent molecular weight. Results described herein suggest that D cyclin, CDK, PCNA and p21 exist in a quaternary complex, that many combinatorial variations of the components (e.g., cyclin D1 or D3 and CDK2, CDK4 and CDK5) assemble in vivo and that each of the resulting quaternary complexes may have a subtly different role in the cell cycle or in different cell types.

Applicant's work, thus, links a human D cyclin whose biochemical function is unknown and which appears to be a G1 cyclin which is identical to a putative oncogene (PRAD1) with a DNA replication and repair factor. Thus, Applicant's work also provides the first biochemical indication of a possible function of D-type cyclins (i.e., as modulators of PCNA function) and, for the first time, provides evidence of a role for D-type cyclins in G1 or S phase of the cell cycle. In addition, Applicant has shown that D-type cyclins are differentially expressed among various cell types and are also differentially expressed or regulated within the same type of cells, depending on the differentiation state of the cells. Therefore, methods of the present invention offer the particular advantage of flexibility and specificity, in that D-type cyclin activity can be altered on the basis of type of D-type cyclin (e.g., D1, D2, D3), across cell types, in a cell-type specific manner or on the basis of cell cycle phase or stage. Further, because of the key roles cyclins have been shown to have in cell cycle control and the evidence, provided herein, of a role for D-type cyclins in the G1 to S transition, the present work provides the basis for a method of regulating the cell cycle which extends to a wide variety of proliferative disorders (any such disorders in which a D-type cyclin plays a determinative role in regulating cell cycle start). Disorders in which the method of the present invention can be used to inhibit cell proliferation include leukemia and tumorigenesis.

Each of the components of the D-type cyclin-containing complex represented in FIG. 16 is a potential target for the present method of altering, particularly inhibiting, cell cycle start and, thus, altering cell division. Selection of the proper "target" constituent of the complex makes the present method highly specific, if desired.

Applicant's work, thus, provides the basis for a better understanding of D-type cyclins, their roles and interactions with other molecules in cell cycle start and approaches to altering or modulating (decreasing or enhancing) eukaryotic cell division, particularly human cell division.

The following is a description of the identification and characterization of human D-type cyclins and of the uses of these novel cyclins and related products, the identification and characterization of a novel cyclin dependent kinase (CDK5); evidence of a role for D-type cyclins in G1 or S phase of the cell cycle; and the discovery that D-type cyclin is associated with three additional polypeptides (CDK, PCNA and p21) in what appears to be a quaternary complex in which many combinatorial variations are possible, resulting in a variety of resulting complexes which may play different roles in the cell cycle or in different cell types.

Isolation and Characterization of Human Cyclin D1, D2 and D3

As represented schematically in FIG. 1 and described in detail in Example 1, a mutant yeast strain in which two of the three CLN genes (CLN1 and CLN2) were inactive and expression of the third was conditional, was used to identify human cDNA clones which rescue yeast from CLN deficiency. A human glioblastoma cDNA library carried in a yeast expression vector (pADNS) was introduced into the mutant yeast strain. Two yeast transformants (pCYCD1-21 and pCYCD1-19) which grew despite the lack of function of all three CLN genes and were not revertants, were identified and recovered in E. coli. Both rescued the mutant (CLN deficient) strain when reintroduced into yeast, although rescue was inefficient and the rescued strain grew relatively poorly.

pCYCD1-19 and pCYCD1-21 were shown, by restriction mapping and partial DNA sequence analysis, to be independent clones representing the same gene. A HeLa cDNA library was screened for a full length cDNA clone, using the 1.2 kb insert of pCYCD1-21 as probe. Complete sequencing was done of the longest of nine positive clones identified in this manner (pCYCD1-H12; 1325 bp). The sequence of the 1.2 kb insert is presented in FIG. 2; the predicted protein product of the gene is of approxiate molecular weight 34,000 daltons.

Cyclin D2 and cyclin D3 cDNAs were isolated using the polymerase chain reaction and three oligonucleotide probes derived from three highly conserved regions of D-type cyclins, as described in Example 4. As described, two 5' oligonucleotides and one 3' degenerate oligonucleotide were used for this purpose. The nucleotide and amino acid sequences of the CCND2 gene and encoded D2 cyclin protein are represented in FIG. 3 and of the CCND3 gene and encoded D3 cyclin protein are represented in FIG. 4. A deposit of plasmid pCYC-D3 was made with the American Type Culture Collection (Rockville, Md.) on May 14, 1991, under the terms of the Budapest Treaty. Accession number 68620 has been assigned to the deposit.

Comparison of the CYCD1-H12-encoded protein sequence with that of known cyclins (see FIG. 5A) showed that there was homology between the new cyclin and A, B and CLN type cyclins, but also made it clear that CYCD1 differs from these existing classes.

An assessment of how this new cyclin gene and its product might be related in an evolutionary sense to other cyclin genes was carried out by a comprehensive comparison of the amino acid sequences of all known cyclins (FIG. 5B and Example 1). Results of this comparison showed that CYCD1 represents a new class of cyclin, designated herein cyclin D.

Expression of cyclin D1 gene in human cells was studied using Northern analysis, as described in Example 2. Results showed that levels of cyclin D1 expression were very low in several cell lines. The entire coding region of the CYCD1 gene was used to probe poly(A)+ RNA from HeLa cells and demonstrated the presence of two major transcripts, one approximately 4.8 kb and the other approximately 1.7 kb, with the higher molecular weight form being the more abundant. Most of the cDNA clones isolated from various cDNA libraries proved to be very similar to clone λCYCD1-H12 and, thus, it appears that the 1.7 kb transcript detected in Northern blots corresponds to the nucleotide sequence of FIG. 2. The origin of the larger (4.8 kb) transcript was unclear. As described in Example 2, it appears that the two mRNAs detected (4.8 kb and 1.7 kb) arose by differential polyadenylation of CYCD1 (FIG. 6).

Differential expression of cyclin D1 in different tissues and cell lines was also assessed, as described in Example 3. Screening of cDNA libraries to obtain full length CYCD1 clones had demonstrated that the cDNA library from the human glioblastoma cell line (U118 MG) used to produce yeast transformants produced many more positives than the other three cDNA libraries (human HeLa cell cDNA, human T cell cDNA, human teratocarcinoma cell cDNA). Northern and Western blotting were carried out to determine whether cyclin D1 is differentially expressed. Results showed (Example 3) that the level of transcript is 7 to 10 fold higher in the glioblastoma (U118 MG) cells than in HeLa cells, and that in both HeLa and U118 MG cells, the high and low molecular weight transcripts occurred. Western blotting using anti-CYL1 antibody readily detected the presence of a 34 kd polypeptide in the glioblastoma cells and demonstrated that the protein is far less abundant in HeLa cells and not detectable in the 293 cells. The molecular weight of the anti-CYCL1 cross-reactive material identified in U118 MG and HeLa cells is exactly that of the human CYCD1 protein expressed in E. coli. Thus, results demonstrated differential occurrence of the cyclin D1 in the cell types analyzed, with the highest levels being in cells of neural origin.

As also described herein (Example 6), human genomic libraries were screened using cDNA probes and genomic clones of human D-type cyclins, specifically D1, D2 and 3, have been isolated and characterized. Nucleic acid sequences of cyclin D1, D2 and D3 promoters are represented in FIGS. 11-13. Specifically, the entire 1.3 kb cyclin D1 CDNA clone was used as a probe to screen a normal human liver genomic library, resulting in identification of three positive clones. One of these clones (G6) contained a DNA insert shown to contain 1150 bp of upstream promoter sequence and a 198 bp exon, followed by an intron. Lambda genomic clones corresponding to the human cyclin D2 and lambda genomic clones corresponding to the human cyclin D3 were also isolated and characterized, using a similar approach. One clone (λD2-G4) was shown to contain (FIG. 8B) a 2.7 kb SacI SmaI fragment which includes 1620 bp of sequence 5' to the presumptive initiating methionine codon identified in D2 cDNA (FIG. 3) and a 195 bp exon followed by a 907 bp intervening sequence. One clone (G9) was shown to contain (FIG. 8C) 1.8 kb of sequence 5' to the presumptive initiating methionine codon identified in D3 cDNA (FIG. 4), a 198 bp exon 1, a 684 bp exon 2 and a 870 bp intron.

Thus, as a result of the work described herein, a novel class of mammalian cyclins, designated cyclin D or D-type cyclin, has been identified and shown to be distinct, on the basis of structure of the gene (protein) product, from previously-identified cyclins. Three members of this new class, designated cyclin D1 or CCND1, cyclin D2 or CCND2 and cyclin D3 or CCND3, have been isolated and sequenced. They have been shown to fulfill the role of another cyclin (CLN type) in activation of-the protein kinase (CDC28) which is essential for cell cycle start in yeast. It has also been shown that the cyclin D1 gene is expressed differentially in different cell types, with expression being highest in cells of neural origin.

Identification of a Novel Kinase Associated with D-Type Cyclins and Demonstration that D-type DNA Replication and Repair Factor PCNA

As described in Example 8, a novel cyclin-dependent kinase, designated CDK5, has been identified, characterized and sequenced. As described briefly below and in detail in Example 8, a human CDNA clone coding for a polypeptide which cross-reacts with antiserum raised against S. pombe p34^(cdc) was isolated. The cDNA encodes a 334 Kd polypeptide which shares 56.8% and 60.3% homology with human CDC2 and CDK2, respectively. The protein was shown, as also described in Example 8, to complex with human cyclin D1 and D3. Based on these findings, the gene product of this clone is designated CDK5. The nucleotide sequence of the human CDK5 cDNA and the amino acid sequence of the encoded protein are shown in FIG. 14.

As also described below and in Example 8, immunological procedures have been used to establish that D-type cyclins associate, in eukaryotic cells, with a variety of potential catalytic subunits (e.g., CDKs, such as CDK2, CDK4 and CDK5). In addition, these procedures have shown that the D-type cyclin and CDK associate with the replication factor PCNA and a polypeptide of 21 kDa apparent molecular weight. The various pair-wise interactions possible are summarized in FIG. 16A.

Human cyclin D1 has been associated with a wide variety of proliferative diseases, but its biochemical role is unknown. As described herein, in human diploid cells, specifically human diploid fibroblasts, cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kDa and 36 kDa are identified in association with cyclin D1. As described in Example 8, it has been shown that the 36 kDa protein is the proliferating cell nuclear antigen, PCNA. PCNA has been described as an essential accessory factor to the delta polymerase, which is required for leading-strand DNA replication and DNA repair. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA, as shown herein. It is proposed that there exists a quaternary complex of D cyclin CDK, PCNA and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4 and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well characterized DNA replication and repair factor.

Investigation of Proteins that Associate with Cyclin D

It is well established that the cdc2-cyclin B protein kinase plays a critical role in controlling the G2/M transition in both mammalian and yeast cells (Draetta, G., Trends. Biol. Sci. 15:378-383 (1990)).

Considerable effort has also been directed toward the isolation of mammalian cyclins that might function at the G1 and S phases of the cell cycle. In a search for a putative oncogene located on band q13 of human chromosome 11, the site of the BCL1 rearrangement in certain lymphomas and leukemias, and also of gene amplification in 15-20% human breast cancers, a cyclin (PRAD1) was identified as the putative oncogene (Motokura et al., Nature 350:512-515 (1991); Withers et al., Mol. Cell. Biol. 11:4846-4853 (1991)). The identical gene, called-cyclin D1, in addition to two further human cyclin genes, cyclin C and cyclin E, were isolated by virtue of their ability to rescue a budding yeast strain that lacks G1 cyclin (Cln) function (Xiong et al., Current Biology 1:362-364 (1991); Koff et al., Cell 66:1217-1228 (1991); Lew et al., Cell 66:1197-1206 (1991)), reviewed in Xiong and Beach, Current Biology 1:362-364 (1991)). In yet another approach, three mouse homologs of human cyclin D1, named CYL1, 2 and 3 were identified as cellular genes whose expression is stimulated by CSF-1 (colony-stimulating factor 1) in macrophage cell lines (Matsushime et al., Cell 65:701-713 (1991)). Two additional human D-type cyclins, cyclin D2 and D3, were isolated as human homologs of murine cyl2 and cyl3 using PCR and low stringency hybridization techniques (Inaba et al., Genomics 13:565-574 (1992); Xiong et al., Genomics 13:575-584 (1992)).

Several lines of very indirect evidence suggest a G1 or S phase role for D-type cyclins. Following stimulation of murine macrophages CSF-1, the levels of both cyclin D1/cyl1 and cyclin D2/cyl2 mRNA increased in the early or middle G1 phase and reached a maximum of the G1/S border. The level of cyclin D/cyl1 protein also increases throughout G1, declines during S and G2 and reaches a nadir after mitosis (Matsushime et al., Cell 65:701-713 (1991); Kiyokawa et al., Proc. Natl. Acad. Sci. USA 89:2444-2447 (1992)). In similar experiments carried out with human diploid fibroblasts, the level of both cyclin D1 and D3 mRNA increases gradually throughout G1 and peaks prior to the onset of S phase. Despite these observations, however, there has been no direct evidence for a G1/S function of any of the D-type cyclins. Applicant has investigated proteins that associate with cyclin D and found a substantial physical association between D cyclins and a DNA replication factor (i.e., PCNA).

Multiple Cyclin D1-Associated Proteins

To identify proteins that specifically associate with cyclin D, anti-cyclin D1 immunoprecipitates of ³⁵ S! methionine-labelled WI38 human diploid fibroblasts lysates were examined (see Example 8, Experimental Procedures). WI38 cells were initially chosen for this study because they are a relatively normal cell line that expresses reasonably high levels of cyclin D1 and a low level of cyclin D3 mRNA (Won et al., Proc. Natl. Acad. Sci. (1992). Human 293 transformed primary embryonal kidney cells were used as controls because they express all three D cyclin mRNAs and proteins at extremely low levels (Xiong, et al., Cell 65:691-699 (1991); FIG. 1A, lane 5). W138 cells express a readily detectable 35 kDa polypeptide that can be immunoprecipitated by the anti-cyclin D1 antiserum. The identity of the 35 kDa protein as cyclin D1 was confirmed by comparison of an immunoprecipitate of the same W138 cell lysate with pre-immune serum (FIG. 1A, lane 4), and with a similar precipitation of 293 cell lysate with the same anti-cyclin D1 antiserum (FIG. 1A, lane 2). Because of the existence of three closely related cyclin D genes in human cells, and weak cross-reactivity of the anti-cyclin D1 antibody to other cyclin D proteins, the identity of the 35 kDa band was further investigated by partial proteolytic mapping. S. aureus V8 partial proteolysis of the 35 kDa band revealed the same pattern as that of similar cleaved cyclin D1 synthesized in vitro, but not as that of cyclin D2 or D3.

In addition to the intense 35 kDa band corresponding to cyclin D1, three other major bands, p33 and p21 and one minor band, p31, appeared specifically in the anti-cyclin D1 precipitates (FIG. 1A, lane 5; FIG. 1C, lane 4). These polypeptides are absent from precipitates of W138 cell lysate using pre-immune serum (FIG. 1A, lane 4) or precipitates of 293 cell lysates with the same anti-cyclin D1 antibody (FIG. 1A, lane 2). The possibility that any of these four bands, in particular p31 and p33, might be cyclin D2 or D3 was ruled out by comparing their partial V8 proteolysis patterns with those of in vitro translated D2 and D3. Precipitation of these polypeptides with anti-cyclin D1 serum is also not likely due to the presence of cross-reactive epitopes in any of these proteins, since they were not detected following immunoprecipitation coupled with Western blotting using the same antibody. Experiments to identify the cyclin D1-associated proteins are described below.

CKD5 Associates with D-Type Cyclins

It has been previously reported that murine macrophages cyclin D1/cyl1 associates with a polypeptide that cross-reacts with an antibody to full-length p34cdc2 of Schizosaccharomyces pombe (G8), but not with an antibody prepared against the C-terminus of human p34cdc2 (Draetta et al,. Cell 50, 319-325 (1987); Draetta and Beach, Cell 54,17-26. (1988); Matsushime et al., Cell 65,701-713 (1991). Essentially identical results were obtained in human W138 cells, suggesting that cyclin D1 associates with a relative of human CDC2.

The G8 antibody was used to screen human cDNA expression libraries (see Experimental Procedures), in order to isolate putative D-type cyclin-associated kinases. Thirty four G8-positive cDNA clones were identified from a HeLa cell cDNA library. Among these, 17 clones encoded CDC2 and another 14 encoded for CDK2. One of the remaining clones encodes an ORF of 292 amino acid residues with a predicted molecular weight of 33,283 daltons. This clone is designated CDK5, since it shares extensive amino acid identity to the known cyclin-dependent kinases (CDKs), including S. pombe CDC2 (53.4%), S. cerevisiae CDC28 (55.9%), human CDC2 (56.8%), and human CDK2 (60.3%), and associates with human D-type cyclins (see below). CDK5 has an inferred amino-acid sequence that is almost identical to a putative protein kinase which was recently identified using polymerase chain reaction (PCR) with primers that are conserved among cdc2 genes (Meyerson et al., EMBO J. 11, 2909-2917 (1992)). CDK5 encodes a sequence of DLKKYFD at amino acid sequence 86 to 92 and the protein referred to as PSSALRE (Meyerson et al., EMBO J. 11:2090-2917 (1992)) contains DLK-NFD at the corresponding region. It is not known whether these two polypeptides are derived from two genes, spliced differently, or whether the discrepancy might have arisen from a cloning or sequencing artifact. In the corresponding region, human CDC2 has the sequence of DLKKYLD and CDK2 has DLKKFMD.

To determine whether CDK5 associates with D cyclins, an antiserum was raised against a peptide corresponding to the unique carboxy-terminal region of CDK5 (see Example 8, Experimental Procedures). This serum does not cross react with human CDC2, CDK2, or CDK4. Immunoprecipitation (FIG. 1C, lane 2) or Western-blotting following immunoprecipitation showed that this antiserum detected a polypeptide with a M_(r) 31 kDa (p31) from cell lysate, which comigrated with CDK5 polypeptide synthesized in vitro and was effectively competed away by the CDK5 antigenic peptide (FIG. 1C, lane 3). The identity of the 31 kDa protein precipitated by the anti-CDK5 antibody was further confirmed to be CDK5 by comparing the partial V8 proteolytic mapping of p31 with in vitro translated CDK5.

Immunoprecipitation of cell lysates of ³⁵ S-methionine labeled W138 cells using the anti-CDK5 antiserum revealed several polypeptides, in addition to p31^(CDK5). Among these, polypeptides of 36 kDa (p36), p35 kDa (p35), 33 kDa (p33) and 21 kDa (p21, FIG. 1C, lane 2) were most prominent and specifically coprecipitated by the anti-CDK5 antiserum. All four polypeptides were absent from precipitates with the pre-immune serum or in the presence of excess amount of the CDK5 carboxy-terminal peptide (FIG. 1C, lanes 1 and 3).

The electrophoretic mobilities of p35 and p33 were found to be the same as that of in vitro translated human cyclin D1 and D3, respectively. To directly test the possibility that the CDK5-associated p35 might correspond to cyclin D1, CDK5 immunoprecipitates were blotted with anti-cyclin D1 antisera. A 35 kDa polypeptide, which comigrated with p35^(cycline) D1, was detected by the anti-cyclin D1 antiserum. Reciprocal blotting of anti-cyclin D1 immunocomplexes by the CDK5 antiserum also revealed the presence of a 31 kDa polypeptide which had the same mobility as p31^(CDK5). Similarly, CDK5 has also been detected in anti-cyclin D3 immunoprecipitates. These data suggest that the CDK5-associated p35 is cyclin D1 and CDK5-associated p33 is cyclin D3.

To seek conclusive evidence of the identity of the CDK5-associated p35 and p33 proteins, partial proteolytic mapping was employed (Cleveland et al., J. Biol. Chem. 252:1102-1106 (1977)). ³⁵ S-labelled p35 purified from anti-CDK5 immunoprecipitates was subjected to partial S. aureus V8 protease digestion and compared with similarly treated human p35^(cyclinD1) obtained either from in vitro translation or from an anti-cyclin D1 immunoprecipitation. The V8 proteolytic pattern of p35 from anti-CDK5 immunoprecipitates was identical to that of cyclin D1, but distinct from that of cyclin D3. Similar experiments were also performed to confirm the identity of p33. The partial proteolytic pattern of the CDK5-associated p33 is identical to that of an vitro translated human cyclin D3, but not D1. Conversely, it has also been determined that the partial V8 digestion pattern of the cyclin D1-associated p31 (FIG. 1A, lane 5 and FIG. 1C, lane 4) is identical to CDK5 obtained either from in vitro translation or anti-CDK5 immunoprecipitation.

CDK2 Associates with Cyclin D

The apparent molecular weight of the cyclin D1-associated p33 (FIG. 1A, lanes 4 and 5) and also the cross reactivity of p33^(CDK2) with the G8 antibody suggests the possibility that p33 might be CDK2. To test this, anti-CDK2 precipitate of a ³⁵ S! methionine-labelled WI38 cell lysate was compared with an anti-cyclin D1 precipitate (FIG. 1A, lanes 5 and 6). As expected, the anti-C terminal CDK2 serum precipitated a 33 kDA protein which was confirmed to be p33^(CDK2) by comparing the partial S. aureus V8 proteolysis pattern of the 33 kDa band with that of in vitro translated CDK2. p33^(CDK2) comigrated with the p33 present in the anti-cyclin D1 precipitate. Reciprocally, anti-CDK2 antiserum also precipitated a 35 kDa protein which comigrated with cyclin D1 (FIG. 1A, lanes 5 and 5).

To seek further evidence for the existence of a possible association between CDK2 and cyclin D1, a WI38 cell lysate was immunoprecipitated with anti-cyclin D1, separated on SDS-PAGE and immunoblotted with anti-CDK2 antiserum. The anti-CDK2 antibody was raised against a carboxy-terminal peptide (Pagano et al., EMBO J. 11:961-971 (1992b)) and its specificity was checked by immuno-blotting bacterially expressed human CDC2, CDK2, CDK3, CDK4 and CDK5. Only CDK2, and not the other four CDK proteins, was recognized by this antibody. CDK2 protein was detected in the precipitate with anti-CDK2 and anti-cyclin D1, but not in that with pre-immune serum nor with anti-CDK2 pre-incubated with competing antigenic peptides. In a reciprocal Western blot experiment, cell lysate was immunoprecipitated with anti-CDK2 and blotted with anti-cyclin D1. Cyclin D1 was detected in the anti-cyclin D1 and anti-CDK2 immunoprecipitates, but not in precipitates with either preimmune serum or anti-CDK2 antiserum pre-incubated with a competing CDK2 peptide.

To test whether CDK2 also associates with cyclin D3, immunoprecipitates using antiserum to the C-terminal peptide of human cyclin D3 (see Example 8, Experimental Procedures) were blotted with anti-CDK2 antiserum. CDK2 was weakly detected in the anti-cyclin D3 precipitate, but not in the control precipitate with anti-cyclin D3 anti-serum pre-incubated with a competing antigen peptide.

Finally, to further confirm the association between CDK2 and cyclin D, partial proteolytic mapping experiments were conducted. Initially, attempts were made to proteolytically map the cyclin D1-associated p33 to compare it with CDK2. However, because of the comigration of CDK2 with yet another predominant protein kinase in the anti-cyclin D1 precipitates, a different proteolytic pattern was obtained. Therefore, the converse experiment was performed. The 35 kDa band in anti-CDK2 immunoprecipitates was excised from SDS-polyacrylamide gel, partially digested with V8 protease and electrophoretically separated and compared with V8 digested p35^(cyclin) D1 derived either from in vitro translation or from an anti-cyclin D1 immunoprecipitation. The pattern of proteolytic cleavage was the same in each case.

pSK-J3/CDK4 is the Predominant p33 Protein Associated with Cyclin D1

The difference in the proteolytic pattern of cyclin D1-associated p33 from that of CDK2 suggested that the majority of D1-associated p33 corresponds to a protein other than CDK2. During attempts to identify this protein, it was suggested to us by Dr. Charles Sherr (St. Jude Children's Research Hospital, Tennessee) that a protein kinase called PSK-J3, originally identified in a screen with mixed oligonucleotide probes derived from conserved regions of serine/threonine kinases (Hanks, S. K., Proc. Natl. Acad. Sci. USA 84:388-392 (1987)), may have cyclin D binding properties. The predicted molecular mass of PSK-J3 is 34 kDa, close to that of p33. Because of its association with D cyclins, as demonstrated below, PSK-J3 is referred to hereinafter as CDK4. In vitro translated CDK4, and that precipitated from a cell lysate with anti-CDK4 serum, showed the same electrophoretic mobility as CDK2 and the D1-associated p33 (FIG. 1B, lanes 2 and 3). The identify of CDK4 precipitated by the anti-CDK4 antiserum was confirmed by comparing its partial V8 mapping pattern to that of in vitro translated CDK4.

Immunoprecipitation-Western blotting experiments were carried out to directly test whether the cyclin D1-associated p33 is CDK4. An anti-CDK4 serum reacted with a 33 kDa protein present in anti-cyclin D1 immunoprecipitates that has the same mobility as the CDK4 precipitated by anti-CDK4, but did not react with precipitates of either CDK2 or CDK5. Reciprocally, the anti-CDK4 antiserum also precipitated a 35 kDa protein detected by anti-cyclin D1 antibody. To further confirm the identity of the cyclin D1-associated p33, the partial V8 digestion pattern of p33 was compared to that of immunoprecipitated CDK4 and CDK2. The cyclin D1-associated p33 displayed a very similar pattern to that of CDK4, but was quite dissimilar to that of CDK2. This result indicates that CDK4 is considerably more abundant (at least as crudely assayed by methionine labelling) than CDK2 in anti-cyclin D1 precipitates of WI38 cells. Similarly, a 33 kDa polypeptide (p33) seen in anti-CDK4 immunoprecipitate has been identified to be cyclin D3 by partial V8 peptide mapping.

Association of p21 with Cyclin D1 and CDK2

In ³⁵ S! methionine-labelled WI38 lysate precipitated with anti-cyclin D1 serum, a 21 kDa protein (p21) appeared to associate specifically with cyclin D1 (FIG. 1). p21 was not present in the precipitates with pre-immune serum (FIG. 1A, lane 4; FIG. 1B, lane 1), nor in the anti-cyclin D1 precipitate derived from 293 cells which contains undetectable levels of cyclin D1 (FIG. 1A, lane 2). Specific association of p21 with cyclin D1 was further supported by the presence of a comigrating 21 kDA protein in immunoprecipitates with sera against CDK2 (FIG. 1A, lane 6), CDK4 (FIG. 1B, lane 3) and CDK5 (FIG. 1C, lane 2). If anti-CDK2 antiserum was pre-blocked with a competing CDK2 peptide, the p21 band, and also p33^(CDK2) and p35^(cyclin) D1 were not seen. Similarly, p21 was also absent from anti-CDK5 immunoprecipitates if the antiserum was pre-incubated with the CDK5 carboxy-terminal antigen peptide (FIG. 1C, lane 3). p21 was not recognized in Western blots by any of the anti-CDK or anti-cyclin D antibodies used in this study. Furthermore, although the total immunoprecipitable CDK2 in 293 cells is similar to that in WI38 cells (FIG. 1A, lanes 3 and 6), the p21 band was not present in the CDK2 immunoprecipitates from 293 cell lysates. This finding suggests that the association of CDK2 and p21 is dependent on cyclin D.

To determine whether the p21 from cyclin D1 immunoprecipitates and CDK2 immunoprecipitates correspond to the same polypeptide, the partial V8 proteolytic pattern of the p21 purified from each source were compared. They are indeed the same. The p21 precipitated by anti-CDK5 anti-serum was also found to be the same as cyclin D1-associated p21. The p21 in the anti-CDK4 immunoprecipitation was also proteolytically mapped (FIG. 1B, lane 3). It gave an identical pattern to the cyclin D1-associated p21. p21 does not correspond to the human max protein or p2^(ras), as its electrophoretic mobility is faster than that of either and it was not recognized by an anti-human ras antibody on Western blots. The molecular identity of p21 is presently unknown.

Cyclin D1-Associated p36 is PCNA

Cyclin D1 precipitates of WI38 cells show associated polypeptides of 21 kDa, 31 kDa and 33 kDa and also a prominent protein of 36 kDa (FIG. 1A, lane 5). p36 was not detected in control precipitates, using either pre-immune serum (FIG. 1A, lane 4; FIG. 1B, lane 1) or in 293 lysates (FIG. 1A, lane 2). A 36 kDa protein, in a lower abundance was also detected in CDK2 (FIG. 1A, lane 6), CDK4 (FIG. 1B, lane 3) and CDK5 (FIG. 1C, lane 2) immunoprecipitates, but not in the precipitates with antiserum pre-incubated with competing peptides (FIG. 1C, lane 3).

While attempting to establish the identity of the p36, four observations suggested the possibility that it might be the human proliferating nuclear antigen, PCNA. First, in an asynchronous population of proliferating WI38 cells, cyclin D1 was predominantly a nuclear protein (data not shown), although the distribution is not identical to the speckled pattern of PCNA (Bravo, R. and H. MacDonald-Bravo, EMBO J., 4:655-661 (1985); Madsen, P. and J. E. Celis, FEBS Lett., 193:5-11 (1985). Second, while the level of cyclin D1 is relatively constant in mitogenically activated WI38 cells, the p36 in ³⁵ S! methionine-labelled cyclin D1 immunoprecipitates was low in quiescent cells and increased at 10-14 hours after stimulation. Ten to fourteen hours after serum stimulation, many WI38 cells are in the late G1, a time which coincides with the onset of PCNA synthesis in serum-stimulated 3T3 fibroblasts (Bravo, R. and H. MacDonald-Bravo, EMBO J., 3:3177-3181 (1984); Celis, J. E. and A. Celis, Proc. Natl. Acad. Sci., USA, 82:3262-3268 (1985); Madsen P. and J. E. Celis, FEBS Lett., 193:5-11 (1985). Third, the apparent molecular weight of p36 is similar to that of PCNA (FIG. 1C, lanes 4 and 5). Finally, anti-PCNA antibody precipitated a 35 kDa polypeptide whose electrophoretic mobility is similar to that of p35^(cyclin) D1 (FIG. 1C, lanes 4 and 5). The identify of the p36 precipitated by the anti-PCNA antibody has been confirmed as PCNA by comparing its V8 peptide map to that of in vitro translated PCNA.

Immunoprecipitation-Western blot experiments were carried out to directly test the possibility that p36 is PCNA. PCNA was readily detected in anti-cyclin D1, cyclin D3, CDK2 and CDK5 immunoprecipitates, but not in the respective control precipitates. In a reciprocal experiment, cyclin D1 and CDK2 were also detected in anti-PCNA immunoprecipitates. It has not been possible to convincingly detect cyclin D3 or CDK5 in PCNA precipitates, possibly due to the low abundance of both proteins in WI38 cells and the relatively poor sensitivity of the D3 and CDK5 antisera in Western blots.

To further assess the similarity between the PCNA and the p36 polypeptide associated with cyclin D1 and CDK2, p36 bands were purified from cyclin D1 and CDK2 immunoprecipitates, separated on SDS-PAGE and their partial V8 proteolytic mapping pattern was compared with that of PCNA. Digestion of cyclin D1-associated p36 by V8 protease revealed the same pattern as that of PCNA derived from anti-PCNA immunoprecipitates and in vitro translated PCNA. Similarly, the digestion patterns of CDK2- and CDK5-associated p36 also match to that of PCNA. The p36 associated with cyclin D1 is PCNA. In addition, proteolytic mapping of the p21 seen in anti-PCNA immunoprecipitate (FIG. 1C, lane 5) showed it to be the same as cyclin D1-associated p21.

Although the experimental techniques used in this study do not formally allow a distinction between the existence of multiple pair-wise interactions between each protein, the data are most simply explained if D cyclin, PCNA, CDK and p21 form a quaternary complex, as illustrated (FIG. 16B). As judged by the intensity of the methionine-labelled bands in the immunoprecipitation reactions, not all the cyclin D is present in the complex (FIG. 1), nor is all the PCNA (FIGS. 1 and 6). However, the relative intensity of the p36 (PCNA), p33 (CDK4) and p21 bands in an anti-cyclin D precipitate is very similar (FIG. 1A, lane 5; FIG. 1B, lane 2; FIG. 1C, lane 4). The results presented herein do not rule out the possibility that cyclin D, with or without the associated proteins described here, might associate with additional partners in vivo. In particular, two polypeptides that migrate either side of the 97 KD molecular weight marker are apparent in anti-cyclin D precipitation reaction (FIG. 1C, lane 4).

PCNA has been described as an essential accessory factor to the delta polymerase, that is required both for leading-strand DNA replication and also for DNA repair (Prelich, G. et al., Nature, 326:517-520 (1987); Prelich G. and B. Stillman, Cell, 53:117-126 (1988); Toschi, L. and R. Bravo, J. Cell Biol. 107:1623-1628 (1988); M. K. K. Shiviji, et al., Cell, 69:367-374 (1992). It localizes in the nucleus at sites of active DNA synthesis and the localization of PCNA, but not its synthesis, is dependent on DNA synthesis. The present studies do not address the specific role of the cyclin D, CDK, PCNA or p21 interactions. It was not possible to detect phosphorylation of any of the respective subunits in in vitro kinase reactions, suggesting that neither PCNA nor p21 is a primary substrate of cyclin D/CDK. Whether cyclin D might be having an activating or inhibitory effect on PCNA functions remains to be determined.

The cyclin D/CDK enzymes that associate with PCNA and p21 might assemble in vivo into a more elaborate multi-protein-DNA synthetic complex, one component of which might be the physiological substrate of cyclin D/CDK. PCNA has generally been biochemically purified from cells in a monomeric form that is unassociated with other proteins (Prelich, G. et al., Nature 346:760-763 (1987)). It is possible that the multi-protein complexes described in the present study were over-looked because they do not comprise the majority of the cellular PCNA. Alternatively, it is possible that PCNA has further non-DNA synthetic cell cycle regulatory roles that have not previously been described and that involve cyclin D and CDK proteins. However, the present studies do provide the first biochemical indication of a possible function of D-type cyclins, as modulators of PCNA function.

As represented in FIG. 16B, the present data are most simply explained if there exists, in vivo, a quaternary cyclin D-p21-CDK-PCNA complex. In addition, there are at least three known human D-type cyclins (Inaba, T. et al., Genomics 13:565-574 (1992); Xiong, Y. et al., Genomics 13:575-584 (1992)) and apparently at least three cyclin D-associated catalytic subunits (CDK2, 4, and 5). All three cyclin D1-associated kinase catalytic subunits, CDK2, CDK4 and CDK5 also associate with cyclin D3. These findings raise the interesting possibility that each of the potential variants of the quaternary complex illustrated in FIG. 16B might exist in vivo. Each might have a subtly different role in the cell cycle or in different cell types.

Uses of the Invention

It is possible, using the methods and materials described herein, to identify genes (DNA or RNA) which encode other cyclins (DNA or RNA which replaces a gene essential for cell cycle start). This method can be used to identify additional members of the cyclin D class or other (non-D type) cyclins of either human or nonhuman origin. This can be done, for example, by screening other cDNA libraries using the budding yeast strain conditional for CLN cyclin expression, described in Example 1, or another mutant in which the ability of a gene to replace cyclin expression can be assessed and used to identify cyclin homologues. This method is carried out as described herein, particularly in Example 1 and as represented in FIG. 1. A cDNA library carried in an appropriate yeast vector (e.g., pADNS) is introduced into a mutant yeast strain, such as the strain described herein (Example 1 and Experimental Procedures). The strain used contains altered CLN genes. In the case of the specific strain described herein, insertional mutations in the CLN1 and CLN2 genes rendered them inactive and alteration of the CLN3 gene allowed for its conditional expression from a galactose-inducible, glucose-repressible promoter; as exemplified, this promoter is a galactose-inducible, glucose-repressible promoter but others can be used.

Mutant yeast transformed with the cDNA library in the expression vector are screened for their ability to grow on glucose-containing medium. In medium containing galactose, the CLN3 gene is expressed and cell viability is maintained, despite the absence of CLN1 and CLN2. In medium containing glucose, all CLN function is lost and the yeast cells arrest in the G1 phase of the cell cycle. Thus, the ability of a yeast transformant to grow on glucose-containing medium is an indication of the presence in the transformant of DNA able to replace the function of a gene essential for cell cycle start. Although not required, this can be confirmed by use of an expression vector, such as pADNS, which contains a selectable marker (the LEU2 marker is present in pADNS). Assessment of the plasmid stability shows whether the ability to grow on glucose-containing medium is the result of reversion or the presence of DNA function (introduction of DNA which replaces the unexpressed or nonfunctional yeast gene(s) essential for cell cycle start). Using this method, cyclins of all types (D type, non-D type) can be identified by their ability to replace CLN3 function when transformants are grown on glucose.

Screening of additional cDNA or genomic libraries to identify other cyclin genes can be carried out using all or a portion of the human D-type cyclin DNAs disclosed herein as probes; for example, all or a portion of the D1, D2 or D3 cDNA sequences of FIGS. 2-4, respectively, or all or a portion of the corresponding genomic sequences described herein can be used as probes. The hybridization conditions can be varied as desired and, as a result, the sequences identified will be of greater or lesser complementarity to the probe sequence (i.e., if higher or lower stringency conditions are used). Additionally, an anti-D type cyclin antibody, such as CYL1 or another raised against D1 or D3 or other human D-type cyclin, can be used to detect other recombinant D-type cyclins produced in appropriate host cells transformed with a vector containing DNA thought to encode a cyclin.

The cyclin-dependent kinase, designated CDK5 and DNA encoding CDK5 are also available as a result of the work described herein. CDK5 has been shown to co-precipitate with D-type cyclin, PCNA and p21 and it is proposed herein that they form a quaternary complex which has a role in vivo in the cell cycle. If this is the case, CDK5 function and/or association with other members of the complex can be altered (enhanced or decreased) in much the same manner as described above for the D-type cyclin. If CDK5 is prevented from binding to D-type cyclin, kinase activation will be prevented. This can be effected as described below. Formation of CDK5-containing quaternary-complexes can also be prevented or enhanced, as can formation of complexes containing other CDKs.

Based on work described herein, it is possible to detect altered expression of a D-type cyclin or increased rates of cell division in cells obtained from a tissue or biological sample, such as blood, urine, feces, mucous or saliva. This has potential for use for diagnostic and prognostic purposes since, for example, there appears to be a link between alteration of a cyclin gene expression and cellular transformation or abnormal cell proliferation. For example, several previous reports have suggested the oncogenic potential of altered human cyclin A function. The human cyclin A gene was found to be a target for hepatitis B virus integration in a hepatocellular carcinoma (Wand, J. et al., Nature 343:555-557 (1990)). Cyclin A has also been shown to associate with adenovirus E1A in virally infected cells (Giordano, A. et al., Cell 58:981-990 (1989); Pines, J. and T. Hunter, Nature 346:760-763 (1990)). Further, the PRAD1 gene, which has the same sequence as the cyclin D1 gene, may play an important role in the development of various tumors (e.g., non-parathyroid neoplasia, human breast carcinomas and squamous cell carcinomas) with abnormalities in chromosome 11q13. In particular, identification of CCND1 (PRAD1) as a candidate BCL1 oncogene provides the most direct evidence for the oncogenic potential of cyclin genes. This also suggests that other members of the D-type cyclin family may be involved in oncogenesis. In this context, the chromosomal locations of the CCND2 and CCND3 genes have been mapped to 12p13 and 6p21, respectively. Region 12p13 contains sites of several translocations that are associated with specific immunophenotypes of disease, such as acute lymphoblastic leukemia, chronic myelomoncytic leukemia, and acute myeloid leukemia. Particularly, the isochromosome of the short arm of chromosome 12 1(12p)! is one of a few known consistent chromosomal abnormalities in human solid tumors and is seen in approximately 90% of adult testicular germ cell tumors. Region 6p21, on the other hand, has been implicated in the manifestation of chronic lymphoproliferative disorder and leiomyoma. Region tp21, the locus of HLA complex, is also one of the best characterized regions of the human genome. Many diseases have been previously linked to the HLA complex, but the etiology of few of these diseases is fully understood. Molecular cloning and chromosomal localization of cyclins D2 and D3 should make it possible to determine whether they are directly involved in these translocations, and if so, whether they are activated. If they prove to be involved, diagnostic and therapeutic methods described herein can be used to assess an individual's disease state or probability of developing a condition associated with or caused by such translocations, to monitor therapy effectiveness (by assessing the effect of a drug or drugs on cell proliferation) and to provide treatment.

The present invention includes a diagnostic method to detect altered expression of a cyclin gene, such as cyclin D1, D2, D3 or another D-type cyclin. The method can be carried out to detect altered expression in cells or in a biological sample. As shown herein, there is high sequence similarity among cyclin D genes, which indicates that different members of D-type cyclins may use similar mechanisms in regulating the cell cycle (e.g., association with the same catalytic subunit and acting upon the same substrates). The fact that there is cell-type-specific differential expression, in both mouse and human cells, makes it reasonable to suggest that different cell lineages or different tissues may use different D-type cyclins to perform very similar functions and that altered tissue-specific expression of cyclin D genes as a result of translocation or other mutational events may contribute to abnormal cell proliferation. As described herein, cyclin D1 is expressed differentially in tissues analyzed; in particular, it has been shown to be expressed at the highest levels in cells of neural origin (e.g., glioblastoma cells). Other D-type cyclins are also expressed differentially among various cell types and further, are differentially expressed even within the same type of cells, depending on the differentiation state. For example, cyclin D2 is differentially expressed in two different T-cell lines which represent distinct stages of T-cell differentiation. In addition, Applicant has shown that expression of D-type cyclin genes correlates with the state of cell growth using human diploid fibroblasts. Thus, differential diagnosis is also possible, in that the type of D cyclin whose function is altered can be determined and a therapeutic agent or drug targeted to that D-type cyclin can be administered, resulting in selective treatment. For example, in those instances where cyclin D1 function is altered, which might be the case in oncogenesis (e.g., in some leukemias and solid tumors), altered cyclin D1 function can be detected and treatment instituted accordingly. This can take the form of administration of a therapeutic agent which specifically inhibits cyclin D1 activity and, thus, specifically inhibits further cell division in those cells in which cyclin D1 is the controlling D-type cyclin. It is possible to combine a drug which specifically alters cyclin D1 function with another agent, such as an antibody, to further target cells in which the anti-cyclin D1 drug is to have its effect. For example an anti-cyclin D1 drug (e.g., an antibody which binds cyclin D1, a peptide which mimics a peptide to which cyclin D1 normally binds) can be attached to a targeting molecule, such as an antibody specific for a marker, such as a cell surface receptor, on cells in which cyclin D1 activity is to be altered. The resulting anti-cyclin D1 drug-targeting molecule conjugate provides specificity in two ways: it delivers an anti-cyclin D1 drug to a specific cell type or types. It is also possible to detect altered D-type cyclin expression and function in a generic sense as well (e.g., to detect all D-type cyclins or a combination of two or more selected D-type cyclins whose altered functions are associated with a condition or disease to be diagnosed).

As a result of the work described herein, D-type cyclin expression can be detected and/or quantitated and results used as an indicator of normal or abnormal (e.g., abnormally high rate of) cell division. Differential expression (either expression in various cell types or of one or more of the types of D cyclins) can also be determined.

In a diagnostic method of the present invention, cells obtained from an individual are processed in order to render nucleic acid sequences in them available for hybridization with complementary nucleic acid sequences. All or a portion of the D1, D2 and/or D3 cyclin (or other D-type cyclin gene) sequences can be used as a probe(s). Such probes can be a portion of a D-type cyclin gene; such a portion must be of sufficient length to hybridize to complementary sequences in a sample and remain hybridized under the conditions used and will generally be at least six nucleotides long. Hybridization is detected using known techniques (e.g., measurement of labeled hybridization complexes, if radiolabeled or fluorescently labeled oligonucleotide probed are used). The extent to which hybridization occurs is quantitated; increased levels of the D-type cyclin gene is indicative of increased potential for cell division.

Alternatively, the extent to which a D-type cyclin (or cyclins) is present in cells, in a specific cell type or in a body fluid can be determined using known techniques and an antibody specific for the D-type cyclin(s). In a third type of diagnostic method, complex formation between the D-type cyclin and the protein kinase with which it normally or typically complexes is assessed, using exogenous substrate, such as histone Hl, as a substrate. Arion, D. et al., Cell, 55:371-378 (1988). In each diagnostic method, comparison of results obtained from cells or a body fluid being analyzed with results obtained from an appropriate control (e.g., cells of the same type known to have normal D-type cyclin levels and/or activity or the same body fluid obtained from an individual known to have normal D-type cyclin levels and/or activity) is carried out. Increased D-type cyclin levels and/or activity may be indicative of an increased probability of abnormal cell proliferation or oncogenesis or of the actual occurrence of abnormal proliferation or oncogenesis. It is also possible to detect more than one type of cyclin (e.g., A, B, and/or D) in a cell or tissue sample by using a set of probes (e.g., a set of nucleic acid probes or a set of antibodies), the members of which each recognize and bind to a selected cyclin and collectively provide information about two or more cyclins in the tissues or cells analyzed. Such probes are also the subject of the present invention; they will generally be detectably labelled (e.g., with a radioactive label, a fluorescent material, biotin or another member of a binding pair or an enzyme).

A method of inhibiting cell division, particularly cell division which would otherwise occur at an abnormally high rate, is also possible. For example, increased cell division is reduced or prevented by introducing into cells a drug or other agent which can block, directly or indirectly, formation of the protein kinase-D type cyclin complex and, thus, block activation of the enzyme. In one embodiment, complex formation is prevented in an indirect manner, such as by preventing transcription and/or translation of the D-type cyclin DNA and/or RNA. This can be carried out by introducing antisense oligonucleotides into cells, in which they hybridize to the cyclin-encoding nucleic acid sequences, preventing their further processing. It is also possible to inhibit expression of the cyclin by interfering with an essential D-type transcription factor. There are reasons to believe that the regulation of cyclin gene transcription may play an important role in regulating the cell cycle and cell growth and oscillations of cyclin mRNA levels are critical in controlling cell division. The G1 phase is the time at which cells commit to a new round of division in response to external and internal sequences and, thus, transcription factors which regulate expression of G1 cyclins are surely important in controlling cell proliferation. Modulation of the transcription factors is one route by which D-type cyclin activity can be influenced, resulting, in the case of inhibition or prevention of function of the transcription factor(s), in reduced D-type cyclin activity. Alternatively, complex formation can be prevented indirectly by degrading the D-type cyclin(s), such as by introducing a protease or substance which enhances cyclin breakdown into cells. In either case, the effect is indirect in that less D-type cyclin is available than would otherwise be the case.

In another embodiment, protein kinase-D type cyclin complex formation is prevented in a more direct manner by, for example, introducing into cells a drug or other agent which binds the protein kinase or the D-type cyclin or otherwise interferes with the physical association between the cyclin and the protein kinase it activates (e.g., by intercalation) or disrupts the catalytic activity of the enzyme. This can be effected by means of antibodies which bind the kinase or the cyclin or a peptide or low molecular weight organic compound which, like the endogenous D-type cyclin, binds the protein kinase, but whose binding does not result in activation of the enzyme or results in its being disabled or degraded. Peptides and small organic compounds to be used for this purpose can be designed, based on analysis of the amino acid sequences of D-type cyclins, to include residues necessary for binding and to exclude residues whose presence results in activation. This can be done, for example, by systematically mapping the binding site(s) and designing molecules which recognize or otherwise associate with the site(s) necessary for activation, but do not cause activation. As described herein, there is differential expression in tissues of D-type cyclins. Thus, it is possible to selectively decrease mitotic capability of cells by the use of an agent (e.g., an antibody or anti-sense or other nucleic acid molecule) which is designed to interfere with (inhibit) the activity and/or level of expression of a selected type (or types) of D cyclin. For example, in treating tumors involving the central nervous system or other non-hematopoietic tissues, agents which selectively inhibit cyclin D1 might be expected to be particularly useful, since D1 has been shown to be differentially expressed (expressed at particularly high levels in cells of neural origin).

Formation of complexes of D-type cyclin, CDK, PCNA and p21 can also be prevented in a similar manner as that described above for inhibiting protein kinase D-type cyclin complex formation. That is, complex formation can be prevented directly (e.g., by means of a drug or agent which binds a component of the complex or otherwise interferes with the physical association of complex components. Complex formation can also be prevented in an indirect manner, such as by preventing transcription and/or translation of DNA and/or RNA encoding a component of the complex, in a similar manner to that described above for blocking D-type cyclin--protein kinase complex formation. Alternatively, complex formation can be prevented indirectly by degrading one or more of its constituents.

Direct inhibition of complex formation can be effected in a variety of ways. Because of the fact that D-type cyclins are differentially expressed in different cell types and at various stages in the cell cycle and that there are numerous combinatorial variations of the quaternary cyclin-containing complex, inhibition can be specific in nature and the agent or drug used can be selected to inhibit the cell cycle (cell proliferation) in a particular cell type and/or at a particular phase of proliferation. For example, a drug which selectively inhibits cyclin D1 can be used to inhibit proliferation of cells (e.g., cells of neural origin) in which it is expressed at high levels.

Alternatively, a drug which selectively inhibits cyclin D2 or cyclin D3 function or its ability to form a quaternary complex can be used. Each of the other complex constituents is also a target whose function or availability for complex formation can be altered. For example, CDK2, CDK4, CDK5 and other cyclin dependent kinases which complex with a D-type cyclin can be inhibited or enhanced, either in terms of their function or their availability for incorporation into the quaternary complex. Drugs or agents which alter PCNA function or availability and drugs or agents which alter p21 function or availability can also be used to inhibit or enhance cell division. In the case of each quaternary complex constituent, it is possible to introduce into cells an agent, such as a small peptide or other organic molecule, which mimics the complex constituent in terms of binding but lacks its active region(s), which results in formation of complexes lacking the activity or interactions of the normally-produced complex.

Direct inhibition of complex formation can also be nonspecific (i.e., can affect the majority of cells or all cells in which the D-type cyclin-containing quaternary complex is formed). This can be done, for example, by introducing into cells a drug which inhibits function or availability of a common component of the quaternary complex (e.g., PCNA) or by introducing a mixture or cocktail of drugs, which together inhibit all D-type cyclins.

Alternatively, indirect inhibition of quaternary complex is possible. That is, a drug or agent which acts to cause less of a complex constituent (e.g., D-type cyclin, CDK, PCNA or p21) available can be used. Such drugs or agents include those, such as anti-sense oligonucleotides, which block transcription or translation and those, such as an enzyme, which degrade complex constituents, either prior to or after their incorporation into a quaternary complex.

Drugs or agents useful in the present method of altering, particularly inhibiting, cell cycle start and, thus, cell division, can be existing compounds or molecules (e.g., small organic molecules, anti-sense oligonucleotides, and inorganic substances) or materials designed for use in the present method. In either case, such drugs can be identified by the method of the present invention.

Once an appropriate drug or agent has been identified, it can be administered to an individual, particularly a human or other vertebrate, by any route effective in introducing the drug or agent into cells in sufficient quantity to have the desired effect (i.e., alteration of cell division). For example, a selected drug can be administered intravenously, intramuscularly, by direct injection into a tumor, via the gastrointestinal tract (e.g., orally), intraperitoneally or intranasally. In some cases, ex vivo administration is appropriate (e.g., in instances where blood or bone marrow is removed from the body, treated and returned to the body).

Generally, the drug or agent used to alter cell division will be included in a formation which can also include a physiological carrier (e.g., a buffer or physiological saline), stabilizers, an adjuvant, and flavoring agents. The quantity of the drug to be administered can be determined empirically and will vary depending on considerations such as the age, weight and height of the recipient and the severity of the condition to be treated.

Antibodies specifically reactive with D-type cyclins of the present invention can also be produced, using known methods. For example, anti-D type cyclin antisera can be produced by injecting an appropriate host (e.g., rabbits, mice, rats, pigs) with the D-type cyclin against which anti sera is desired and withdrawing blood from the host animal after sufficient time for antibodies to have been formed. Monoclonal antibodies can also be produced using known techniques. Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989); Hallow, E. and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York (1988). Antibodies specifically reactive with CDK5, can also be produced using known methods. The present invention also includes a method of screening compounds or molecules for their ability to inhibit or suppress the function of a cyclin, particularly a D-type cyclin. For example, mutant cells as described herein, in which a D-type cyclin such as D1 or D3, is expressed, can be used. A compound or molecule to be assessed for its ability to inhibit a D-type cyclin is contacted with the cells, under conditions appropriate for entry of the compound or molecule into the cells. Inhibition of the cyclin will result in arrest of the cells or a reduced rate of cell division. Comparison of the rate or extent of cell division in the presence of the compound or molecule being assessed with cell division of an appropriate control (e.g., the same type of cells without added test drug) will demonstrate the ability or inability of the compound or molecule to inhibit the cyclin. Existing compounds or molecules (e.g., those present in a fermentation broth or a chemical "library") or those developed to inhibit the cyclin activation of its protein kinase can be screened for their effectiveness using this method. Drugs which inhibit D-type cyclin are also the subject of this invention.

The present invention also includes a method of screening compounds or molecules for their ability to alter formation of the quaternary complex described herein. This method is carried out in much the same way as the method, described above, for identifying compounds or molecules which inhibit a D-type cyclin. In the subject method, the compound or molecule to be tested and cells in which D-type cyclin-containing complex is formed are combined, under conditions appropriate for complex formation to occur and entry into cells of the compound or molecule being tested. Complex formation can be determined, as described herein. Inhibition of a complex constituent or of complex formation will result in arrest of the cells or a reduced rate of cell division. Comparison of the rate or extent of cell division in the presence of the compound or molecule being tested with the rate or extent in the absence of the compound or molecule will demonstrate whether it has an effect on cell division (i.e., division to a lesser extent in the presence of the compound or molecule tested than in its absence is an indication the compound or molecule is an inhibitor). Drugs or agents which inhibit complex formation and, as a result, cell division, are also the subject of this invention.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES

Experimental procedures for Examples 1-3 are presented after Example 3.

Example 1 Identification of Human cDNA Clones that Rescue CLN Deficiency

In S. cerevisiae, there are three C1n proteins. Disruption of any one CLN gene has little effect on growth, but if all three CLN genes are disrupted, the cells arrest in G1 (Richardson, H. E. et al., Cell 59:1127-1133 (1989)). A yeast strain was constructed, as described below, which contained insertional mutations in the CLN1 and CLN2 genes to render them inactive. The remaining CLN3 gene was further altered to allow for conditional expression from the galactose-inducible, glucose-repressible promoter GAL1 (see FIG. 1). The strain is designated 305-15d #21. In medium containing galactose the CLN3 gene is expressed and despite the absence of both CLN1 and CLN2, cell viability is retained (FIG. 1). In a medium containing glucose, all CLN function is lost and the cells arrest in the G1 phase of the cell cycle.

A human glioblastoma cDNA library carried in the yeast expression vector pADNS (Colicelli, J. et al., Pro. Natl. Acad. Sci. USA 86:3599-3603 (1989)) was introduced into the yeast. The vector pADNS has the LEU2 marker, the 2μ replication origin, and the promoter and terminator sequences from the yeast alcohol dehydrogenase gene (FIG. 1). Approximately 3×10⁶ transformants were screened for the ability to grow on glucose containing medium. After 12 days of incubation, twelve colonies were obtained. The majority of these proved to be revertants. However, in two cases, the ability to grow on glucose correlated with the maintenance of the LEU2 marker as assessed by plasmid stability tests. These two yeast transformants carried plasmids designated pCYCD1-21 and CYCD1-19 (see below). Both were recovered in E. coli. Upon reintroduction into yeast, the plasmids rescued the CLN deficient strain, although the rescue was inefficient and the rescued strain grew relatively poorly.

The restriction map and partial DNA sequence analysis revealed that pCYCD1-19 and pCYCD1-21 were independent clones representing the same gene. The 1.2 kb insert of pCYCD1-21 was used as probe to screen a human HeLa cDNA library for a full length cDNA clone. Approximately 2 million cDNA clones were screened and 9 positives were obtained. The longest one of these clones, pCYCD1-H12 (1325 bp), was completely sequenced (FIG. 2). The sequence exhibits a very high GC content within the coding region (61%) and contains a poly A tail (69 A residues). The estimated molecular weight of the predicted protein product of the gene is 33,670 daltons starting from the first in-frame AUG codon at nucleotide 145 (FIG. 2). The predicted protein is related to other cyclins (see below) and has an unusually low pI of 4.9 (compared to 6.4 of human cyclin A, 7.7 of human cyclin B and 5.6 of CLN1), largely contributed by the high concentration of acidic residues at its C-terminus.

There are neither methionine nor stop codons 5' to the predicted initiating methionine at nucleotide 145. Because of this and also because of the apparent N-terminal truncation of CYCD1 with respect to other cyclins (see below for more detail), four additional human cDNA libraries were further screened to see if the λCYCD1-H12 clone might lack the full 5' region of the cDNA. Among more than 100 cDNA clones isolated from these screens, none was found that had a more extensive 5' region than that of λCYCD1-H12. The full length coding capacity of clone H12 was later confirmed by Western blot analysis (see below).

CYCD1 encodes the smallest (34 kd) cyclin protein identified so far, compared to the 49 kd human cyclin A, 50 kd human cyclin B and 62 kd S. cerevisiae CLN1. By comparison with A and B type cyclins, the difference is due to the lack of almost the entire N-terminal segment that contains the so called "destruction box" identified in both A and B type cyclins (Glotzer, M. et al., Nature 349:132-138 (1991)).

Sequence Analysis of D1 and Comparison with Other Cyclins

Sequence analysis revealed homology between the CYCD1-H12 encoded protein and other cyclins. However, it is clear that CYCD1 differs from the three existing classes of cyclins, A, B and CLN. To examine how this new cyclin gene might be evolutionary related to other cyclins, a comprehensive amino acid sequence comparison of all cyclin genes was conducted. Fifteen previously published cyclin sequences as well as CYCD1 were first aligned using a strategy described in detail by Xiong and Eickbush (Xiong, Y. and T. H. Eickbush, EMBO J. 9:3353-3362 (1990)). Effort was made to reach the maximum similarity between sequences with the minimum introduction of insertion/deletions and to include as much sequence as possible. With the exception of CLN cyclins, this alignment contains about 200 amino acids residues which occupies more than 70% of total coding region of CYCD1 (FIG. 5A). There is a conserved domain and some scattered similarities between members of A and B type cyclins N-terminal to the aligned region (Glotzer, M. et al., Nature 349:132-138 (1991)), but this is not present in either CLN cyclins or CYCD1 and CYL1 and so they were not included in the alignment.

The percent divergence for all pairwise comparisons of the 17 aligned sequences was calculated and used to construct an evolutionary tree of cyclin gene family using the Neighbor-Joining method (Saitou, N. and M. Nei, Mol. Biol. Evol. 4:406-425 (1987) and Experimental Procedures). Because of the lowest similarity of CLN cyclins to the other three classes, the tree (FIG. 5B) was rooted at the connection between the CLN cyclins and the others. It is very clear from this evolutionary tree that CYCD1, CYCD2 and CYCD3 represent a distinct new class of cyclin, designated cyclin D.

Example 2 Expression of the Cyclin D1 Gene in Human Cells

Expression of cyclin D1 gene in human cells was studied by Northern analysis. Initial studies indicated that the level of cyclin D1 expression was very low in several cell lines. Poly (A)+RNA was prepared from HeLa cells and probed with the entire coding region of CYCD1 gene. Two major transcripts of 4.8 kb and 1.7 kb were detected. The high molecular weight form was the most abundant. With the exception of a few cDNA clones, which were truncated at either the 5' or 3' ends, most of the cDNA clones isolated from various different cDNA libraries are very similar to the clone λCYCD1-H12 (FIG. 2). Thus, it appears that the 1.7 kb transcript detected in Northern blots corresponds to nucleotide sequence in FIG. 2.

To understand the origin of the larger 4.8 kb transcript, both 5' and 3' end sub-fragments of the λCYCD1-H12 clone were used to screen both cDNA and genomic libraries, to test whether there might be alternative transcription initiation, polyadenylation and/or mRNA splicing. Two longer cDNA clones, λCYCD1-H034 (1.7 kb) from HeLa cells and λDYDC1-T078 (4.1 kb) from human teratocarcinoma cells, as well as several genomic clones were isolated and partially sequenced. Both λCYCD1-H034 and λCYCD1-T078 have identical sequences to λCYCD1-H12 clone from their 5' ends (FIG. 6). Both differ from λCYCD1-H12 in having additional sequences at the 3' end, after the site of polyadenylation. These 3' sequences are the same in λCYCD1-H034 and λCYCD1-T078, but extend further in the latter clone (FIG. 6). Nucleotide sequencing of a genomic clone within this region revealed colinearity between the cDNAs and the genomic DNA (FIG. 6). There is a single base deletion (an A residue) in λCYCD1-T078 cDNA clone. This may be the result of polymorphism, although it is not possible to exclude the possibility that some other mechanism is involved. The same 4.8 kb transcript, but not the 1.7 kb transcript, was detected using the 3' end extra fragment from clone T078 as a probe.

It appears that the two mRNAs detected in Northern blots arise by differential polyadenylation (FIG. 6). Strangely, there is no recognizable polyadenylation sequence (AAUAAA) anywhere within the sequence of clone λCYCD1-H12, even though polyadenylation has clearly occurred (FIG. 2). There is also no close variant of AAUAAA (nothing with less than two mismatches).

Example 3 Differential Expression of Cyclin D1 Gene in Different Cell Types

During the screening of cDNA libraries to obtain full length clones of CYCD1, it became evident that the cDNA library derived from the human glioblastoma cell line (U118 MG) from which the yeast transformants were obtained gave rise to many more positives than the other four cDNA libraries. Northern and Western blotting were carried out to explore the possibility that cyclin D1 might be differentially expressed in different tissues or cell lines. Total RNA was isolated from U118 MG cells and analyzed by Northern blot using the CYCD1 gene coding region as probe. The level of transcript is 7 to 10 fold higher in the glioblastoma cells, compared to HeLa cells. In both HeLa and U118 MG cells, both high and low molecular weight transcripts are observed.

To investigate whether the abundant CYCD1 message in the U118 MG cell line is reflected at the protein level, cell extracts were prepared and Western blotting was performed using anti-CYL1 prepared against mouse CYL1 (provided by Matsushime, H. et al.). This anti-CYL1 anti-body was able to detect nanogram quantities of recombinant CYCD1 on Western blots (data not shown), and was also able to detect CYCD1 in the original yeast transformants by immunoprecipitation and Western analysis. Initial experiments using total cell extracts, from HeLa, 293 or U118 MG cells failed to detect any signal. However, if the cell extracts were immunoprecipitated with the serum before being subjected to SDS-PAGE and immunoblotting, a 34 kd polypeptide was readily detected in U118 MG cells. The protein is far less abundant in HeLa cells and was not detectable in 293 cells. The molecular weight of the anti-CYCL1 crossreactive material from U118 MG and HeLa is exactly that of the human CYCD1 protein expressed in E. coli. This argues that the sequenced cDNA clones contain the entire open reading frame.

EXPERIMENTAL PROCEDURES

Strain Construction

The parental strain was BF305-15d (MATa leu2-3 leu2-112 his3-11 his3-15 ura3-52 trp1 adel met14 arg5,6) (Futcher, B. and J. Carbon, Mol. Cell. Biol. 6:2213-2222 (1986)). The strain was converted into a conditional cln-strain in three steps. First, the chromosomal CLN3 gene was placed under control of the GAL1 promoter. A 0.75 kb EcoRI-BamHI fragment containing the bidirectional GAL10-GAL1 promoters was fused to the 5' end of the CLN3 gene, such that the BamHI (GAL1) end was attached 110 nucleotides upstream of the CLN3 start codon. An EcoRI fragment stretching from the GAL10 promoter to the middle of CLN3 (Nash, R. et al., EMBO J. 7:4335-4346 (1988)) was then subcloned between the XhoI and EcoRI sites of pBF30 (Nash, R. et al., EMBO J. 7:4335-4346 (1988)). The ligation of the XhoI end to the EcoRI end was accomplished by filling in the ends with Klenow, and blunt-end ligating (destroying the EcoRI site). As a result, the GAL1 promoter had replaced the DNA normally found between -110 and -411 upstream of CLN3. Next, an EcoRI to SphI fragment was excised from this new pBF30 derivative. This fragment had extensive 5' and 3' homology to the CLN3 region, but contained the GAL1 promoter and a URA3 marker just upstream of CLN3. Strain BF305-15d was transformed with this fragment and Ura+ transformants were selected. These were checked by Southern analysis. In addition, average cell size was measured when the GAL1 promoter was induced or uninduced. When the GAL1 promoter was induced by growing the cells in 1% raffinose and 1% galactose, mode cell volume was about 25 μm³ (compared to a mode volume of about 40 μm³ for the parental strain) whereas when the promoter was not induced (raffinose alone), or was repressed by the presence of glucose, cell volume was much larger than for the wildtype strain. These experiments showed that CLN3 had been placed under control of the GAL1 promoter. It is important to note that this GAL1-controlled, glucose repressible gene is the only source of CLN3 protein in the cell.

Second, the CLN1 gene was disrupted. A fragment of CLN1 was obtained from I. Fitch, and used to obtain a full length clone of CLN1 by hybridization, and this was sub-cloned into a pUC plasmid. A BamHI fragment carrying the HIS3 gene was inserted into an NcoI site in the CLN1 open reading frame. A large EcoRI fragment with extensive 5' and 3' homology to the CLN1 region was then excised, and used to transform the BF305-15d GAL-CLN3 strain described above. Transformation was done on YNB-his raffinose galactose plates. His+ clones were selected, and checked by Southern analysis.

Finally, the CLN2 gene was disrupted. A fragment of CLN2 was obtained from I. Fitch, and used to obtain a full length clone of CLN2 by hybridization, and this was subcloned into a pUC plasmid. An EcoRI fragment carrying the TRP1 gene was inserted into an SpeI site in the CLN2 open reading frame. A BamHI-KpnI fragment was excised and used to transform the BF305-15d GAL-CLN3 HIS3::cln1 strain described above. Transformation was done on YNB-trp raffinose galactose plates. Trp+ clones were selected. In this case, because the TRP1 fragment included an ARS, many of the transformants contained autonomously replicating plasmid rather than a disrupted CLN2 gene. However, several percent of the transformants were simple TRP1::cln2 disruptants, as shown by phenotypic and Southern analysis.

One particular 305-15d GAL1-CLN3 HIS3::c1n1 TRP1::c1n2 transformant called clone #21 (referred to hereafter as 305-15d #21) was analyzed extensively. When grown in 1% raffinose and 1% galactose, it had a doubling time indistinguishable from the CLN wild-type parental strain. However, it displayed a moderate Wee phenotype (small cell volume), as expected for a CLN3 overexpressor. When glucose was added, or when galactose was removed, cells accumulated in G1 phase, and cell division ceased, though cells continued to increase in mass and volume. After overnight incubation in the G1-arrested state, essentially no budded cells were seen, and a large proportion of the cells had lysed due to their uncontrolled increase in size.

When 305-15d #21 was spread on glucose plates, revertant colonies arose at a frequency of about 10-7. The nature of these glucose-resistant, galactose-independent mutants was not investigated.

Yeast Spheroplasts Transformation

S. cerevisiae spheroplasts transformation was carried out according to Burgers and Percival and Allshire (Burgers, P. M. J. and K. J. Percival, Anal. Biochem. 163:391-397 (1987); Allshire, R. C., Proc. Natl. Acad. Sci. USA 87:40433-4047 (1990)).

Cell Culture

HeLa and 293 cells were cultured at 37-C either on plates or in suspension in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Glioblastoma U118 MG cells were cultured on plates in DMEM supplemented with 15% fetal bovine serum and 0.1 mN non-essential amino acid (GIBCO).

Nucleic Acid Procedures

Most molecular biology techniques were essentially the same as described by Sambrook et al. (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Phagmid vectors pUC118 or pUC119 (Vieira, J. and J. Messing, et al. Meth. Enzymol. 153:3-11 (1987)) or pBlueScript (Stratagene) were used as cloning vectors. DNA sequences were determined either by a chain termination method (Sanger, F. et al. Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) using Sequenase Kit (United States Biochemical) or on an Automated Sequencing System (373A, Applied Biosystems).

Human HeLa cell cDNA library in λZAP II was purchased from Stratagene. Human T cell cDNA library in λgtlO was a gift of M. Gillman (Cold Spring Harbor Laboratory). Human glioblastoma U118 MG and glioblastoma SW1088 cell cDNA libraries in λZAP II were gifts of M. Wigler (Cold Spring Harbor Laboratory). Human teratocarcinoma cell cDNA library λgtlO was a gift of Skowronski (Cold Spring Harbor Laboratory). Normal human liver genomic library λGEM-11 was purchased from Promega.

Total RNA from cell culture was extracted exactly according to Sambrook et al. (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)) using guanidium thiocyanate followed by centrifugation in CsCl solution. Poly(A)+RNA was isolated from total RNA preparation using Poly (A)+Quick push columns (Stratagene). RNA samples were separated on a 1% agaroseformaldehyde-MOPS gel and transferred to a nitrocellulose filter. Northern hybridizations (as well as library screening) were carried out at 68° C. in a solution containing 5×Denhardt's solution, 2×SSC, 0.1% SDS, 100 μg/ml denatured Salmon sperm DNA, 25 μM NaPO₄ (pH7.0) and 10% dextran sulfate. Probes were labelled by the random priming labelling method (Feinberg, A. and B. Vogelstein, Anal. Biochem. 132:6-13 (1983)). A 1.3 kb Hind III fragment of cDNA clone pCYCD1-H12 was used as coding region probe for Northern hybridization and genomic library screening, a 1.7 kb Hind III-EcoRI fragment from cDNA clone pCYCD1-T078 was used as 3' fragment probe.

To express human cyclin D1 gene in bacteria, a 1.3 kb Nco I-Hind II fragment of pCYCD1-H12 containing the entire CYCD1 open reading frame was subcloned into a T7 expression vector (pET3d, Studier, F. W. et al., Methods in Enzymology 185:60-89 (1990)). Induction of E. coli strain BL21 (DE3) harboring the expression construct was according to Studier (Studier, F. W. et al., Methods in Enzymology 185:60-89 (1990)). Bacterial culture was lysed by sonication in a lysis buffer (5 mM EDTA, 10% glycerol, 50 mM Tris-HCL, pH 8.0, 0.005% Triton X-100) containing 6M urea (CYCD1 encoded p34 is only partial soluble in 8M urea), centrifuged for 15 minutes at 20,000 g force. The pellet was washed once in the lysis buffer with 6M urea, pelleted again, resuspended in lysis buffer containing 8M urea, and centrifuged. The supernatant which enriched the 34 kd CYCD1 protein was loaded on a 10% polyacrymide gel. The 34 kd band was cut from the gel and eluted with PBS containing 0.1% SDS.

Sequence Alignment and Formation of an Evolutionary Tree

Protein sequence alignment was conducted virtually by eye according to the methods described and discussed in detail by Xiong and Eickbush (Xiong, Y. and T. H. Eickbush, EMBO J. 9:3353-3362 (1990)). Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion.

Numbers within certain sequences indicate the number of amino acid residues omitted from the sequence as the result of insertion (e.g., for CLN1, . . . TWG25RLS . . . indicates that 25 amino acids have been omitted between G and R). Sources for each sequence used in this alignment and in the construction of an evolutionary tree (FIG. 5B) are as follows: CYCA-Hs, human A type cyclin (Wang, J. et al., Nature 343:555-557 (1990)); CYCA-X1, Xenopus A-type cyclin (Minshull, J. et al., EMBO J. 9:2865-2875 (1990)); CYCA-Ss, clam A-type cyclin (Swenson, R. I. et al., Cell 47:867-870 (1986); CYCA-Dm, Drosophila A-type cyclin (Lehner, C. F. and P. H. O'Farrell, Cell 56:957-968 (1989)); CYCB1-Hs, human B1-type cyclin (Pines, J. and T. Hunter, Cell 58:833-846 (1989)); CYCB1-X1 and CYCB2-X1, Xenopus B1- and B2-type cyclin (Minshull, J. et al., Cell 56:947-956 (1989)); CYCB-Ss, clam B-type cyclin (Westendorf, J. M. et al., J. Cell Biol. 108:1431-1444 (1989)); CYCB-Asp, starfish B-type cyclin (Tachibana, K. et al., Dev. Biol. 140:241-252 (1990)); CYCB-Arp, sea urchin B-type cyclin (Pines, J. and T. Hunter, EMBO J. 6:2987-2995 (1987)); CYCB-Dm, Drosophila B-type cyclin (Lehner, C. F. and P. H. O'Farrell, Cell 61:535-547 (1990)); CDC13-Sp, S. pombe CDC13 (Booher, R. and D. Beach, EMBO J. 7:2321-2327 (1988)); CLN1-Sc and CLN2-Sc, S. cerevisiae cyclin 1 and 2 (Hadwiger, J. A. et al., Proc. Natl. Acad. Sci. USA 86:6255-6259 (1989)); CLN3-Sc, S. cerevisiae cyclin 3 (Nash, R. et al., EMBO J. 7:43354346 (1988)).

A total of 17 cyclin sequences were aligned and two representative sequences from each class are presented in FIG. 5A.

Percent divergence of all pairwise comparison of 17 sequences were calculated from 154 amino acid residues common to all 17 sequences, which does not include the 50 residue segments located at N-terminal part of A, B and D-type cyclins because of its absence from CLN type cyclins. A gap/insertion was counted as one mismatch regardless of its size. Before tree construction, all values were changed to distance with Poisson correction (d=-log_(e) S, where the S=sequence similarity (Nei, M., Molecular Evolutionary Genetics pp. 287-326 Columbia University Press, N.Y. (1987)). Calculation of pairwise comparison and Poisson correction were conducted using computer programs developed at University of Rochester. Evolutionary trees of cyclin gene family was generated by the Neighbor-Joining program (Saitou, N. and M. Nei, Mol. Biol. Evol. 4:406-567 (1987)). All calculations were conducted on VAX computer MicroVMS V4.4 of Cold Spring Harbor Laboratory. The reliability of the tree was evaluated by using a subset sequence (e.g., A, B and D-type cyclins), including more residues (e.g., the 50-residue segment located at C-terminal of A, B and D-type cyclins, FIG. 5A) or adding several other unpublished cyclin sequences. They all gave rise to the tree with the same topology as the one presented in FIG. 5B.

Immunoprecipitation and Western Blots

Cells from 60 to 80% confluent 100 mm dish were lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 0.5% Nadeoxycholate, 1 mM PMSF) for 30 minutes on ice. Immunoprecipitation was carried out using 1 mg protein from each cell lysate at 4° C. for overnight. After equilibrated with the lysis buffer, 60 μl of Protein A-agarose (PIERCE) was added to each immunoprecipitation and incubated at 4° C. for 1 hour with constant rotating. The immunoprecipitate was washed three times with the lysis buffer and final resuspended in 50 μl 2×SDS protein sample buffer, boiled for 5 minutes and loaded onto a 10% polyacrymide gel. Proteins were transferred to a nitrocellulose filter using a SDE Electroblotting System (Millipore) for 45 minutes at a constant current of 400 mA. The filter was blocked for 2 to 6 hours with 1×PBS, 3% BSA and 0.1% sodium azide, washed 10 minutes each time and 6 times with NET gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin and 0.02 sodium azide), radiolabelled with 125I-Protein A for 1 hour in blocking solution with shaking. The blot was then washed 10 minutes each time and 6 times with the NET gel buffer before autoradiography.

The tree was constructed using the Neighbor-Joining method (Saitou, N. and M. Nei, Mol. Biol. Evol., 4:406425 (1987). The length of horizontal line reflects the divergence. The branch length between the node connecting the CLN cyclins and other cyclins was arbitrary divided.

MATERIALS AND METHODS

The following materials and methods were used in the work described in Examples 4-6.

Molecular Cloning

The human HeLa cell cDNA library, the human glioblastoma cell U118 MG cDNA library, the normal human liver genomic library, and the hybridization buffer were the same as those described above. A human hippocampus cDNA library was purchased from Stratagene, Inc. High- and low-stringency hybridizations were carried out at 68° and 50° C., respectively. To prepare template DNA for PCR reactions, approximately 2 million lambda phages from each cDNA library were plated at a density of 10⁵ PFU/150-mm plate, and DNA was prepared from the plate lysate according to Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

Example 4 Isolation of Human Cyclin D2 and D3 cDNAs

To isolate human cyclin D2 and D3 cDNAs, two 5' oligonucleotides and one 3' degenerate oligonucleotide were derived from three highly conserved regions of human CCND1, mouse cyl1, cyl2, and cyl3 D-type cyclins (Matsushime, H. et al., Cell 65:701-713 (1991); Xiong, Y. et al., Cell 65:691-699; FIG. 8). The first 5' oligonucleotide primer, HCND11, is a 8192-fold degenerate 38-mer (TGGATG T/C!TNGA A/G!GTNTG T/C!GA A/C!GA A/G!CA A/G!AA A/G!TG T/C!GA A/G!GA) (SEQ ID No. 37), encoding 13 amino acids (WMLEVCEEQKCEE) (SEQ ID No. 38). The second 5' oligonucleotide primer, HCND12, is a 8192-fold degenerate 29-mer (GTNTT T/C!CCN T/C!TNGCNATGAA T/C!TA T/C!TNGA) (SEQ ID No. 39), encoding 10 amino acids (VFPLAMNYLD) (SEQ ID No. 40). The 3' primer, HCND13, is a 3072-fold degenerate 24-mer ( A/G!TCNGT A/G!TA A/G/T!AT A/G!CANA A/G! T/C!TT- . T/C!TC) (SEQ ID No. 41), encoding 8 amino acids (EKLCIYTD) (SEQ ID No. 42). The PCR reactions were carried out for 30 cycles at 94° C. for 1 min, 48° C. for 1 min, and 72° C. for 1 min. The reactions contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM each of dATP, dGTP, dCTP, and dTTP, 2.5 units of Taq polymerase, 5 μM of oligonucleotide, and 2-10 μg of template DNA. PCR products generated by HCND11 and HCND13 were verified in a second-round PCT reaction using HCND12 and HCND13 as the primers. After resolution on a 1.2% agarose gel, DNA fragments with the expected size (200 bp between primer HCND11 and HCND13) were purified and subcloned into the SmaI site of phagmid vector pUC118 for sequencing.

To isolate full-length cyclin D3 cDNA, the 201-bp fragment of the D3 PCR product was labeled with oligonucleotide primers HCND11 and HCND13 using a random-primed labeling technique (Feinberg, A. P. et al., Anal. Biochem. 12:6-13 (1983)) and used to screen a human HeLa cell cDNA library. The probe used to screen the human genomic library for the CCND3 gene was a 2-kb EcoRI fragment derived from cDNA clone λD3-H34. All hybridizations for the screen of human cyclin D3 were carried out at high stringency.

The PCR clones corresponding to CCND1 and CCND3 have been repeatedly isolated from both cDNA libraries; CCND2 has not. To isolate cyclin D2, a 1-kb EcoRI fragment derived from mouse cyl2 cDNA was used as a probe to screen a human genomic library. Under low-stringency conditions, this probe hybridized to both human cyclins D1 and D2. The cyclin D1 clones were eliminated through another hybridization with a human cyclin D1 probe at high stringency. Human CCND2 genomic clones were subsequently identified by partial sequencing and by comparing the predicted protein sequence with that of human cyclins D1 and D3 as well as mouse cyl2.

As described above, human CCND1 (cyclin D1) was isolated by rescuing a triple Cln deficiency mutant of Saccharomyces cerevisiae using a genetic complementation screen. Evolutionary proximity between human and mouse, and the high sequence similarity among cyl1, cyl2, and cyl3, suggested the existence of two additional D-type cyclin genes in the human genome. The PCR technique was first used to isolate the putative human cyclin D2 and D3 genes. Three degenerate oligonucleotide primers were derived from highly conserved regions of human CCND1, mouse cyl1, cyl2, and cyl3. Using these primers, cyclin D1 and a 200-bp DNA fragment that appeared to be the human homolog of mouse cyl3 from both human HeLa cell and glioblastoma cell cDNA libraries was isolated. A human HeLa cell cDNA library was screened with this PCR product as probe to obtain a full-length D3 clone. Some 1.2 million cDNA clones were screened, and six positives were obtained. The longest cDNA clone from this screen, λD3-H34 (1962 bp), was completely sequenced (FIG. 4).

Because a putative human cyclin D2 cDNA was not detected by PCR, mouse cyl2 cDNA was used as a heterologous probe to screen a human cDNA library at low stringency. This resulted, initially, in isolation of 10 clones from the HeLa cell cDNA library, but all corresponded to the human cyclin D1 gene on the basis of restriction mapping. Presumably, this was because cyclin D2 in HeLa cells is expressed at very low levels. Thus, the same probe was used to screen a human genomic library, based on the assumption that the representation of D1 and D2 should be approximately equal. Of the 18 positives obtained, 10 corresponded to human cyclin D1 and 8 appeared to contain human cyclin D2 sequences (see below). A 0.4-kb BamHI restriction fragment derived from λD2-G1 1 of the 8 putative cyclin D2 clones, was then used as probe to screen a human hippocampus cDNA library at high stringency to search for 8 full-length cDNA clone of the cyclin D2 gene. Nine positives were obtained after screening of approximately 1 million cDNA clones. The longest cDNA clone, λD2-P3 (1911 bp), was completely sequenced (FIG. 3). Neither λD2-P3 nor λD3-H34 contains a poly(A) sequence, suggesting that part of the 3' untranslated region might be missing.

The DNA sequence of λD2-P3 revealed an open reading frame that could encode a 289-amino-acid protein with a 33,045-Da calculated molecular weight. A similar analysis of λD3-H34 revealed a 292-amino-acid open reading frame encoding a protein with a 32,482-Da calculated molecular weight. As in the case of human cyclin D1, there is neither methionine nor stop codons 5' to the presumptive initiating methionine codon for both λD2-P3 (nucleotide position 22, FIG. 3) and λD3-H34 (nucleotide position 101, FIG. 4). On the basis of the protein sequence comparison with human cyclin D1 and mouse cyl1 (FIG. 7) and preliminary results of the RNase protection experiment, both λD2-P3 and λD3-H34 are believed to contain full-length coding regions.

The protein sequence of all 11 mammalian cyclins identified to date were compared to assess their structural and evolutionary relationships. This includes cyclin A, cyclins B1 and B2, six D-type cyclins (three from human and three from mouse), and the recently identified cyclins E and C (FIG. 7). Several features concerning D-type cyclins can be seen from this comparison. First, as noted previously for cyclin D1, all three cyclin D genes encode a similar small size protein ranging from 289 to 295 amino acid residues, the shortest cyclins found so far. Second, they all lack the so-called "destruction box" identified in the N-terminus of both A- and B-type cyclins, which targets it for ubiquitin-dependent degradation (Glotzer, M. et al., Nature 349:132-138 (1991)). This suggests either that the D-type cyclins have evolved a different mechanism to govern their periodic degradation during each cell cycle or that they do not undergo such destruction. Third, the three human cyclin D genes share very high similarity over their entire coding region: 60% between D1 and D2, 60% between D2 and D3, and 52% between D1 and D3. Fourth, members of the D-type cyclins are more closely related to each other than are members of the B-type cyclins, averaging 78% for three cyclin D genes in the cyclin box versus 57% for two cyclin B genes. This suggests that the separation (emergence) of D-type cyclins occurred after that of cyclin B1 from B2. Finally, using the well-characterized mitotic B-type cyclin as an index, the most closely related genes are cyclin A (average 51%), followed by the E-type (40%), D-type (29%), and C-type cyclins (20%).

Example 5 Chromosome Localization of CCND2 and CCND3

The chromosome localization of CCND2 and CCND3 was determined by fluorescence in situ hybridization. Chromosome in situ suppression hybridization and in situ hybridization banding were performed as described previously (Lichter, T. et al., Science 247:64-69 (1990); Baldini, A. et al., Genomics 9:770-774 (1991)). Briefly λD2-G4 and λD3-G9 lambda genomic DNAs containing inserts of 15 and 16 kb, respectively, were labeled with biotin-11-dUTP (Sigma) by nick-translation (Brigatti, D. J. et al., Urology 126:32 50 (1983); Boyle, A. L., In Current Protocols in Molecular Biology, Wiley, N.Y., 1991). Probe size ranged between 200 and 400 nucleotides, and unincorporated nucleotides were separated from probes using Sephadex G-50 spin columns (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Metaphase chromosome spreads prepared by the standard technique (Lichter, T. et al., Science 247:64-69 (1990)) were hybridized in situ with biotin-labeled D2-G4 or D3-G9. Denaturation and preannealing of 5 μg of DNase-treated human placental DNA, 7 μg of DNased salmon sperm DNA, and 100 ng of labeled probe were performed before the cocktail was applied to Alu prehybridized slides. The in situ hybridization banding pattern used for chromosome identification and visual localization of the probe was generated by cohybridizing the spreads with 40 ng of an Alu 48-mer oligonucleotide. This Alu oligo was chemically labeled with digoxigenin-11-dUTP (Boehringer-Mannheim) and denatured before being applied to denatured chromosomes. Following 16-18 h of incubation at 37° C. and posthybridization wash, slides were incubated with blocking solution and detection reagent (Lichter, T. et al., Science 247:64-69 (1990)). Biotin-labeled DNA was detected using fluorescence isothiocyanate (FITC)-conjugated avidin DCS (5 μg/ml) (Vector Laboratories); digoxigenin-labeled DNA was detected using a rhodamine-conjugated anti-digoxigenin antibody (Boehringer-Mannheim). Fluorescence signals were imaged separately using a Zeiss Axioskop-20 epifluorescence microscope equipped with a cooled CCD camera (Photometrics CH220). Camera control and image acquisition were performed using an Apple Macintosh IIX computer. The gray scale images were pseudocolored and merged electronically as described previously (Baldini, A. et al., Genomics 9:770-774 (1991)). Image processing was done on a Macintosh IIci computer using Gene Join Maxpix (software by Tim Rand in the laboratory of D. Ward, Yale) to merge FITC and rhodamine images. Photographs were taken directly from the computer monitor.

Chromosomal fluorescence in situ hybridization was used to localize D2-G4 and D3-G9. The cytogenetic location of D2-G4 on chromosome 12p band 13 and that of D3-G9 on chromosome 6p band 21 were determined by direct visualization of the two-color fluorescence in situ hybridization using the biotin-labeled probe and the digoxigen-labeled Alu 48-mer oligonucleotide (FIG. 5).

The Alu 48-mer R-bands, consistent with the conventional R-banding pattern, were imaged and merged with images generated from the D2-G4 and D3-G9 hybridized probes. The loci of D2-G4 and D3-G9 were visualized against the Alu banding by merging the corresponding FITC and rhodamine images. This merged image allows the direct visualization of D2-G4 and D3-G9 on chromosomes 12 and 6, respectively. The D2-G4 probe lies on the positive R-band 12p13, while D3-G9 lies on the positive R-band 6p21. Cross-hybridization was not detected with either pseudo-gene cyclin D2 or D3, presumably because the potentially cross-hybridizing sequence represents only a sufficiently small proportion of the 15- and 16-kb genomic fragments (nonsuppressed) used as probe, and the nucleotide sequences of pseudogenes have diverged from their ancestral active genes.

Example 6 Isolation and Characterization of Genomic Clones of Human D-Type Cyclins

Genomic clones of human D-type cyclins were isolated and characterized to study the genomic structure and to obtain probes for chromosomal mapping. The entire 1.3-kb cyclin D1 cDNA clone was used as probe to screen a normal human liver genomic library. Five million lambda clones were screened, and three positives were obtained. After initial restriction mapping and hybridizations, lambda clone G6 was chosen for further analysis. A 1.7-kb BamHI restriction fragment of λD1-G6 was subcloned into pUC118 and completely sequenced. Comparison with the cDNA clones previously isolated and RNase protection experiment results (Withers, D. A. et al., Mol. Cell. Biol. 11:4846-4853 (1991)) indicated that this fragment corresponds to the 5' part of the cyclin D1 gene. As shown in FIG. 8A, it contains 1150 bp of upstream promoter sequence and a 198-bp exon followed by an intron.

Eighteen lambda genomic clones were isolated from a similar screening using mouse cyl2 cDNA as a probe under low-stringency hybridization conditions, as described above (Example 4). Because it was noted in previous cDNA library screening that the mouse cyl2 cDNA probe can cross-hybridize with the human D1 gene at low stringency, a dot-blot hybridization at high stringency was carried out, using the human D1 cDNA probe. Ten of the 18 clones hybridized with the human D1 probe and 8 did not. On the basis of the restriction digestion analysis, the 8 lambda clones that did not hybridize with the human D1 probe at high stringency fall into three classes represented by λD2-G1, λD2-G2, and λD2-G4, respectively. These three lambda clones were subcloned into a pUC plasmid vector, and small restriction fragments containing coding region were identified by Southern hybridization using a mouse cyl2 cDNA probe. A 0.4-kb BamHI fragment derived from λD2-G1 was subsequently used as a probe to screen a human hippocampus cell cDNA library at high stringency. Detailed restriction mapping and partial sequencing indicated that λD2-G1 and λD2-G2 were two different clones corresponding to the same gene, whereas λD2-G4 appeared to correspond to a different gene. A 2.7-kb SacI-SmaI fragment from λD2-G4 and 1.5-kb BclI-BglII fragment from λD2-G1 have been completely sequenced. Nucleotide sequence comparison revealed that the clone λD2-G4 corresponds to the D2 cDNA clone λD2-P3 (FIG. 3). As shown in FIG. 8A, the 2.7-kb SacI-SmaI fragment contains 1620 bp of sequence 5' to the presumptive initiating methionine codon identified in D2 cDNA (FIG. 3) and a 195-bp exon followed by a 907-bp intervening sequence.

Lambda genomic clones corresponding to the human cyclin D3 were isolated from the same genomic library using human D3 cDNA as a probe. Of four million clones screened, nine were positives. Two classes of clones, represented by λD3-G4 and λD3-G9, were distinguished by restriction digestion analysis. A 2.0-kb HindIII-ScaI restriction fragment from λD3-G5 and a 3.7-kb SacI-HindIII restriction fragment from λD3-G9 were further subcloned into a pUC plasmid vector for more detailed restriction mapping and complete sequencing, as they both hybridized to the 5' cyclin D3 cDNA probe. As presented in FIG. 9C, the 3.7-kb fragment from clone G9 contains 1.8 kb of sequence 5' to the presumptive initiating methionine codon identified in D3 cDNA (FIG. 4), a 198-bp exon 1, a 684-bp exon 2, and a 870-bp intron.

Comparison of the genomic clones of cyclins D1, D2, and D3 revealed that the coding regions of all three human CCND genes are interrupted at the same position by an intron (indicated by an arrow in FIG. 8). This indicated that the intron occurred before the separation of cyclin D genes.

Example 7 Isolation and Characterization of Two Cyclin D Pseudogenes

The 1.5-kb BclI-BglII fragment subcloned from clone λD2-G1 has been completely sequenced and compared with cyclin D2 cDNA clone λD2-P3. As shown in FIG. 10, it contains three internal stop codons (nucleotide positions 495, 956, and 1310, indicated by asterisks), two frame-shifts (position 1188 and 1291, slash lines), one insertion, and one deletion. It has also accumulated many missense nucleotide substitutions, some of which occurred at the positions that are conserved in all cyclins. For example, triplet CGT at position 277 to 279 of D2 cDNA (FIG. 3) encodes amino acid Arg, which is an invariant residue in all cyclins (see FIG. 8). A nucleotide change from C to T at the corresponding position (nucleotide 731) in clone D2-G1 (FIG. 10) gave rise to a triplet TGT encoding Cys instead of Arg. Sequencing of the 2.0-kb HindIII-ScaI fragment from clone λD3-G5 revealed a cyclin D3 pseudogene (FIG. 11). In addition to a nonsense mutation (nucleotide position 1265), two frameshifts (position 1210 and 1679), a 15-bp internal duplication (underlined region from position 1361 to 1376), and many missense mutations, a nucleotide change from A to G at position 1182 resulted in an amino acid change from the presumptive initiating methionine codon ATG to GTG encoding Val. On the basis of these analyses, we conclude that clones λD2-G1 and λD3-G5 contain pseudogenes of cyclins D2 and D3, respectively.

Example 8 Identification of a Cyclin Dependent Kinase and Demonstration that D-Type Cyclins Associated with Multiple Protein Kinases and the DNA Replication and Repair Factor PCNA

Experimental Procedures

Cells

Human diploid lung fibroblast WI38 cells were obtained from American Type Culture Collection at passage 13 and were grown in Dulbecco-Modified Eagle media supplemented with 10% fetal bovine serum and used between passages 16-22. 293 cells were cultured similarly.

Antibodies

To raise anti-cyclin D1 antibody, a 609 bp DNA restriction fragment encoding 202 amino acid residues (˜25 kDa) of human cyclin D1 amino-terminal region (the NCoI fragment from nucleotides 143 to 751 in FIG. 2 of Xiong, et al., 1991) was subcloned into a phage T7 expression vector, pET-3d (Studier, et al., 1990) and introduced into E. coli strain BL21 (DE3). Bacterial extracts were prepared in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5 and 10% glycerol) by disrupting cells with sonication and clarifying the supernatant by centrifugation at 20,000 g for 10 minutes. Pellets containing insoluble cyclin D protein was resuspended in lysis buffer supplemented with 8M urea, after 30 minutes shaking at room temperature, the suspension was centrifuged again at 20,000 g for 10 minutes. Pellets containing insoluble cyclin D protein was resuspended in SDS sample buffer and separated on 10% SDS-polyacrylamide gel. The 25 kDa cyclin D protein was visualized and excised after staining the gel with 0.25M KCl in the cold room. Gel slices were further crushed by repeated passage through an 18 gauge needle and cyclin D protein was extracted by incubating the crushed gel particles with PBS containing 0.1% SDS at 42° C. for several hours and used for injection of rabbits. To affinity purify the anti-cyclin D1 immunoglobulins, bacterially produced p25 proteins were cross-linked to the Reacti-Gel (6×) according to the manufacturer's instruction. The affinity column was washed with excess volume of PBS containing 0.05% Tween-20 before and after crude serum was applied to the column. Bound immunoglobulins were eluted with Glycine-NaCl (pH 2.5) into 1.5 M Tris-HCl, pH 8.5 to instantly neutralize the antibodies. To reduce the high background caused by immunoglobulin proteins, affinity purified anti-cyclin D1 was crosslinked to protein A agarose beads according to Harlow and Lane (1988). On Western blots, the anti-cyclin D1 antiserum weakly cross-reacts with bacterially produced human cyclin D2, very poorly with bacterially produced human cyclin D3, and detects a single band from total WI38 cell lysates. In the immunoprecipitations with RIPA buffer (0.1% SDS), more than 90% of cyclin D1-associated p36, p33, p31 and p21 are disappeared while the amount of cyclin D1 remained to be the same as that in the immunoprecipitations with NP40 (0.5%) buffers.

For anti-CDK5 antibody production a peptide CYFSDFCPP (SEQ ID No. 39) with the underlined amino acid residues corresponding to the carboxy-terminal region of CDK5 was synthesized. The peptide was coupled to keyhole limpet hemocyanin (Pierce) which was then used to immunize rabbits as described (Green, et al., 1982).

Anti-cyclin D3 peptide antibody was similarly raised against a synthetic peptide CDELDOASTPTDVRDIDL (SEQ ID No. 40) with the underlined region corresponding to the carboxy-terminal region of human cyclin D3. The rabbit was later stimulated with bacterial produced full length human cyclin D3. Cyclin D3 specific immunoglobulins were purified on an affinity column in which the 17-mer cyclin D3 peptides were crosslinked to the Reacti-Gel (6×). The affinity purified anti-cyclin D3 peptide antibody does not cross-react with bacterially produced cyclin D1 or D2 on Western blots and does not immunoprecipitate cyclin D1 from W138 cell lysates.

The antiserum against S. pombe p34^(cdc2) (G8) was described before (Draetta, et al., 1987). Human auto-immune anti-PCNA antiserum was from Dr. Michael Mathews (Cold Spring Harbor Laboratory, New York). Affinity purified anti-PCNA monoclonal antibody used in Western-blots was purchased from Boehringer Mannheim. Affinity purified anti-PCNA monoclonal antibody used in immunoprecipitation of FIG. 6B was purchased from oncogene Science. Anti-CDK2 peptide antiserum was a gift of Dr. Giulio Draetta (EMBL, Heidelberg, Pagno, et al., 1992b) and does not cross-react with CDC2, CDK4 and CDK5 polypeptides. Anti-CDK4 antiserum was a gift of Dr. Steven Hanks (Vanderbilt University, Tennessee) and was raised against a fusion protein of glutathione S transferase (GST) and a C-terminal portion of CDK4. It does not cross-react with CDK2 and CDK5.

Screening Human cDNA Expression Library

A human HeLa cell cDNA expression library constructed in lambda ZAP II (#936201) was from Stratagene. Human p34^(cdc2) was highly insoluble when produced from bacteria. The conventional antibody screening method (Young and Davis, 1983) is suitable only when there is sufficient amount of soluble recombinant proteins in phage plaques. The screening method, therefore, was modified to include a step which involved the use of 6M guanidine to solubilize recombinant proteins after they have been transferred to nitrocellulose paper, a procedure which was initially developed to produce refolded recombinant proteins with certain activities (Vinson, et al., 1988). Two million phage plaques from the λZAP II HeLa cDNA library were screened with antiserum against S. pombe p34^(cdc2) (G8). After overlaying phage plaques with IPTG-impregnated nitrocellulose filters for 4 hours at 42° C., the filters were removed from culture dishes and were then treated with 6M guanidine-HCl in a buffer containing 25 mM Hepes, pH 7.0, 50 mM NaCl, 2 mM DTT for 10 min at 25° C. The filters were washed free of guanidine with Tris-buffered saline before antibody incubation. This procedure enhanced our antibody detection signal greatly which probably was due to the solubilization of bacterial-produced poly-peptide precipitates by guanidine. The G8-positive cDNA clones subcloned into pBluescript SK vector (Stratagene) and sequenced from both directions using ABI automated DNA sequencer (Model 373A). For sequence homology search, the FASTA program was used (Pearson and Lipman, 1988).

Immunoprecipitation and Western-Blotting

For metabolic labelling with ³⁵ S! methionine, subconfluent (40-60%) cells were washed twice with prewarmed labelling media (methionine-, cystine-free DMEM ICN! supplemented with 10% dialyzed fetal bovine serum, GIBCO!). After 30 minutes incubation with the labelling media, ³⁵ S! methionine (Trans³⁵ S-label, ICN) was added to media (approximately 200 μCi/ml) and continued to incubate for four to six hours before lysis. All steps of immunoprecipitations were carried out in the cold room. Cells from 40 to 60% confluent 150 mM dish were washed twice with cold PBS and scraped into NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5% NP-40, 1 mM PMSF, 25 μg/ml leupeptin, 25 μ/ml aprotitin, 1 mM benzamidine and 10 μg/ml trypsin inhibitor) and lysed by rotating for 15 to 30 minutes. Nuclei were removed by centrifugation at 15,000 g for 5 minutes and lysates were pre-cleared by incubating with either pre-immune serum or normal rabbit serum and IgG sorb (The Enzyme Center, Inc.) for 20 to 30 minutes followed by a 10 minute centrifugation at 15,000 g. Antibody pre-coupled to the protein A agarose beads (Pierce) was added to the clarified lysates and incubated for six to eight hours. Immunoprecipitates were washed three to four times with lysis buffer at room temperature, resuspended in SDS sample buffer and separated on SDS-polyacrylamide gels.

For the ³⁵ S methionine-labelled precipitates, poly-acrylamide gels (except those for V8 proteolytic mapping experiments) were fixed with 10% glacial acetic acid and 30% methanol for 30 minutes to one hour, enhanced by impregnating with autoradiography enhances (Du Pont) for 30 minutes and precipitated in water for 15 to 30 minutes. Enhanced gels were dried and exposed to X-ray films at -70° C. For Western-blotting, polypeptides were transferred to a nitrocellulose filer using a SDE Electroblotting System (Millipore) for 45 minutes at constant current of 400 mA. The filter was blocked for 1 to 3 hours with TBST (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20) containing 5% dry milk, incubated with primary antibody for 4 hours to overnight in TBST containing 5% dry milk and washed 4 times, 10 minutes each time, with TBST. Appropriate secondary antibody (1:10,000 dilution of either horseradish peroxidase linked sheet anti-mouse Ig or donkey anti-rabbit Ig, Amersham) were incubated with filters for one hour and specific proteins were detected using an enhanced chemiluminescence system (ECL, Amersham).

Partial Proteolytic Peptide Mapping

Human cyclin D1, cyclin D2, cyclin D3, CDC2, CDK2, CDK3, CDK4, CDK5 and PCNA were subcloned into pBluescript vector (Stratagene) for in vitro translation with T7 RNA polymerase using a TNT coupled reticulocyte lysate system (Promega). Immunoprecipitation of ³⁵ S! methionine-labelled lysates and SDS-polyacrylamide gel electrophoresis were the same as described above. Polyacrylamide gels were dried without prior fixation and enhanced treatment, exposed to Fuji image plates and visualized on Fuji bio-imaging analyzer BAS2000. Appropriate protein bands were excised from the gels using image printout as template, in-gel partially digested with various amount of S. aureus V8 protease according to (Cleveland, et al., 1977) and (Harlow and Lane, 1988), separated on a 17.5% SDS-PAGE. Gels were dried and exposed to a X-ray film for 2 weeks, or analyzed on a Fuji image analyzer BAS2000.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 50     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1325 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 145..1029     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     GCAGTAGCAGCGAGCAGCAGAGTCCGCACGCTCCGGCGAGCGCCAGAACAGCGCGAGGGA60     GCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCCTCCCC120     AGCTGCCCAGGAAGAGCCCCAGCCATGGAACACCAGCTCCTGTGCTGCGAA171     MetGluHisGlnLeuLeuCysCysGlu     15     GTGGAAACCATCCGCCGCGCGTACCCCGATGCCAACCTCCTCAACGAC219     ValGluThrIleArgArgAlaTyrProAspAlaAsnLeuLeuAsnAsp     10152025     CGGGTGCTGCGGGCCATGCTGAAGGCGGAGGAGACCTGCGCGCCCTCG267     ArgValLeuArgAlaMetLeuLysAlaGluGluThrCysAlaProSer     303540     GTGTCCTACTTCAAATGTGTGCAGAACGACGTCCTCCCGTCCATGCCG315     ValSerTyrPheLysCysValGlnAsnAspValLeuProSerMetPro     455055     AAGATCGTCGCCACCTGGATGCTGGAGGTCTGCGAGGAACAGAAGTGC363     LysIleValAlaThrTrpMetLeuGluValCysGluGluGlnLysCys     606570     GAGGAGGAGCTCTTCCCGCTGGCCATGAACTACCTGGACCGGTTCCTG411     GluGluGluLeuPheProLeuAlaMetAsnTyrLeuAspArgPheLeu     758085     TCGCTGGAGCCCGTGAAAAAGAGCCGCCTGCAGCTGCTGGGGGCCACT459     SerLeuGluProValLysLysSerArgLeuGlnLeuLeuGlyAlaThr     9095100105     TGCATGTTCGTGGCCTCTAAGATGAAGGAGACCATCCCCCTGACGGCC507     CysMetPheValAlaSerLysMetLysGluThrIleProLeuThrAla     110115120     GAGAAGCTGTGCATCTACACCGACGCCTCCATCCCCCCCGAGGACCTG555     GluLysLeuCysIleTyrThrAspAlaSerIleProProGluAspLeu     125130135     CTGCAAATGGAGCTGCTCCTGGTGAACAAGCTCAAGTGGAACCTGGCC603     LeuGlnMetGluLeuLeuLeuValAsnLysLeuLysTrpAsnLeuAla     140145150     GCAATGACCCCGCACGATTTCATTGAACACTTCCTCTCCAAAATGACA651     AlaMetThrProHisAspPheIleGluHisPheLeuSerLysMetThr     155160165     GAGGCGGAGGAGAACAAACAGATCATCCGCAAACACGCGCAGACCTTC699     GluAlaGluGluAsnLysGlnIleIleArgLysHisAlaGlnThrPhe     170175180185     GTTGCCTCTTGTGCCACAGATCTGAAGTTCATTTCCAATCCGCCCTCC747     ValAlaSerCysAlaThrAspLeuLysPheIleSerAsnProProSer     190195200     ATGGTGGCAGCGGGGACCGTGGTCGCCGCAGTGCAAGGCCTGAACCTG795     MetValAlaAlaGlyThrValValAlaAlaValGlnGlyLeuAsnLeu     205210215     AGGAGCCCCAACAACTTCCTGTCGTACTACCGCCTCACACGCTTCCTC843     ArgSerProAsnAsnPheLeuSerTyrTyrArgLeuThrArgPheLeu     220225230     TCCAGAGTGATCAAGTGTGACCCAGACTGCCTCCGGGCCTCCCAGGAG891     SerArgValIleLysCysAspProAspCysLeuArgAlaSerGlnGlu     235240245     CAGATCGAAGCCCTGCTGGAGTCAAGCCTGCGCCAGGCCCACCAGAAC939     GlnIleGluAlaLeuLeuGluSerSerLeuArgGlnAlaHisGlnAsn     250255260265     ATGGACCCCAAGGCCGCCGAGGAGGAGGAAGAGGAGGAGGAGGAGGTG987     MetAspProLysAlaAlaGluGluGluGluGluGluGluGluGluVal     270275280     GACCTGGCTTGCACACCCACCGACGTCCCGGACCTGGACATC1029     AspLeuAlaCysThrProThrAspValProAspLeuAspIle     285290295     TGAGGGGCCCAGCGAGGCGGGCGCCACCGCCACCCGCAGCGAGGGCGGAGCCGGCCCCAG1089     GTGCTCCACATGACAGTCCCTCCTCTCCGGAGCATTTTGATACCAGAAGGGAAACCTTCA1149     TTCTCCTTGTTGTTGGTTGTTTTTTCCTTTGCTCTTTCCCCCTTCCATCTCTCACTTAAC1209     CAAAACAAAAAGATTACCCAAAAACTGTCTTTAAAAGAGAGAGAGAGAAAAAAAAAAAAA1269     AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA1325     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 295 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     MetGluHisGlnLeuLeuCysCysGluValGluThrIleArgArgAla     151015     TyrProAspAlaAsnLeuLeuAsnAspArgValLeuArgAlaMetLeu     202530     LysAlaGluGluThrCysAlaProSerValSerTyrPheLysCysVal     354045     GlnAsnAspValLeuProSerMetProLysIleValAlaThrTrpMet     505560     LeuGluValCysGluGluGlnLysCysGluGluGluLeuPheProLeu     65707580     AlaMetAsnTyrLeuAspArgPheLeuSerLeuGluProValLysLys     859095     SerArgLeuGlnLeuLeuGlyAlaThrCysMetPheValAlaSerLys     100105110     MetLysGluThrIleProLeuThrAlaGluLysLeuCysIleTyrThr     115120125     AspAlaSerIleProProGluAspLeuLeuGlnMetGluLeuLeuLeu     130135140     ValAsnLysLeuLysTrpAsnLeuAlaAlaMetThrProHisAspPhe     145150155160     IleGluHisPheLeuSerLysMetThrGluAlaGluGluAsnLysGln     165170175     IleIleArgLysHisAlaGlnThrPheValAlaSerCysAlaThrAsp     180185190     LeuLysPheIleSerAsnProProSerMetValAlaAlaGlyThrVal     195200205     ValAlaAlaValGlnGlyLeuAsnLeuArgSerProAsnAsnPheLeu     210215220     SerTyrTyrArgLeuThrArgPheLeuSerArgValIleLysCysAsp     225230235240     ProAspCysLeuArgAlaSerGlnGluGlnIleGluAlaLeuLeuGlu     245250255     SerSerLeuArgGlnAlaHisGlnAsnMetAspProLysAlaAlaGlu     260265270     GluGluGluGluGluGluGluGluValAspLeuAlaCysThrProThr     275280285     AspValProAspLeuAspIle     290295     (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1970 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 22..948     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     GAATTCCCGCCGGGCTTGGCCATGGAGCTGCTGTGCCACGAGGTGGACCCG51     MetGluLeuLeuCysHisGluValAspPro     1510     GTCCGCAGGGCCGTGCGGGACCGCAACCTGCTCGGAGACGACCGCGTC99     ValArgArgAlaValArgAspArgAsnLeuLeuGlyAspAspArgVal     152025     CTGCAGAACCTGCTCACCATCGAATTCCCGCCGGGCTTGGCCATGGAG147     LeuGlnAsnLeuLeuThrIleGluPheProProGlyLeuAlaMetGlu     303540     CTGCTGTGCCACGAGGTGGACCCGGTCCGCAGGGAGGAGCGCTACCTT195     LeuLeuCysHisGluValAspProValArgArgGluGluArgTyrLeu     455055     CCGCAGTGCTCCTACTTCAAGTGCGTGCAGAAGGACATCCAACCCTAC243     ProGlnCysSerTyrPheLysCysValGlnLysAspIleGlnProTyr     606570     ATGCGCAGAATGGTGGCCACCTGGATGCTGGAGGTCTGTGAGGAACAG291     MetArgArgMetValAlaThrTrpMetLeuGluValCysGluGluGln     75808590     AAGTGCGAAGAAGAGGTCTTCCCTCTGGCCATGAATTACCTGGACCGT339     LysCysGluGluGluValPheProLeuAlaMetAsnTyrLeuAspArg     95100105     TTCTTGGCTGGGGTCCCGACTCCGAAGTCCCATCTGCAACTCCTGGGT387     PheLeuAlaGlyValProThrProLysSerHisLeuGlnLeuLeuGly     110115120     GCTGTCTGCATGTTCCTGGCCTCCAAACTCAAAGAGACCAGCCCCCTG435     AlaValCysMetPheLeuAlaSerLysLeuLysGluThrSerProLeu     125130135     ACCGCGGAGAAGCTGTGCATTTACACCGACAACTCCATCAAGCCTCAG483     ThrAlaGluLysLeuCysIleTyrThrAspAsnSerIleLysProGln     140145150     GAGCTGCTGGAGTGGGAACTGGTGGTGCTGGGGAAGTTGAAGTGGAAC531     GluLeuLeuGluTrpGluLeuValValLeuGlyLysLeuLysTrpAsn     155160165170     CTGGCAGCTGTCACTCCTCATGACTTCATTGAGCACATCTTGCGCAAG579     LeuAlaAlaValThrProHisAspPheIleGluHisIleLeuArgLys     175180185     CTGCCCCAGCAGCGGGAGAAGCTGTCTCTGATCCGCAAGCATGCTCAG627     LeuProGlnGlnArgGluLysLeuSerLeuIleArgLysHisAlaGln     190195200     ACCTTCATTGCTCTGTGTGCCACCGACTTTAAGTTTGCCATGTACCCA675     ThrPheIleAlaLeuCysAlaThrAspPheLysPheAlaMetTyrPro     205210215     CCGTCGATGATCGCAACTGGAAGTGTGGGAGCAGCCATCTGTGGGCTC723     ProSerMetIleAlaThrGlySerValGlyAlaAlaIleCysGlyLeu     220225230     CAGCAGGATGAGGAAGTGAGCTCGCTCACTTGTGATGCCCTGACTGAG771     GlnGlnAspGluGluValSerSerLeuThrCysAspAlaLeuThrGlu     235240245250     CTGCTGGCTAAGATCACCAACACAGACGTGGATTGTCTCAAAGCTTGC819     LeuLeuAlaLysIleThrAsnThrAspValAspCysLeuLysAlaCys     255260265     CAGGACCAGATTGAGGCGGTGCTCCTCAATAGCCTGCAGCAGTACCGT867     GlnAspGlnIleGluAlaValLeuLeuAsnSerLeuGlnGlnTyrArg     270275280     CAGGACCAACGTGACGGATCCAAGTCGGAGGATGAACTGGACCAAGCC915     GlnAspGlnArgAspGlySerLysSerGluAspGluLeuAspGlnAla     285290295     AGCACCCCTACAGACGTGCGGGATATCGACCTGTGAGGATGCCAGTTGGGCCG968     SerThrProThrAspValArgAspIleAspLeu     300305     AAAGAGAGAGACGCGTCCATAATCTGGTCTCTTCTTCTTTCTGGTTGTTTTTTTCTTTGT1028     GTTTTAGGGTGAAACTTAAAAAAAAAATTCTGCCCCCACCTAGATCATATTTAAAGATCT1088     TTTAGAAGTGAGAGAAAAAGGTCCTACGAAAACGGAATAATAAAAAGCATTTGGTGCCTA1148     TTTGAAGTACAGCATAAGGGAATCCCTTGTATATGCGAACAGTTATTGTTTGATTATGTA1208     AAAGTAATAGTAAAATGCTTACAGGGAAACCTGCAGAGTAGTTAGAGAATATGTATGCCT1268     GCAATATGGGACCAAATTAGAGGAGACTTTTTTTTTTCATGTTATGAGCTAGCACATACA1328     CCCCCTTGTAGTATAATTTCAAGGAACTGTGTACGCCATTTATCGATGATTAGATTGCAA1388     AGCAATGAACTCAAGAAGGAATTGAAATAAGGAGGGACATGATGGGGAAGGAGTACAAAA1448     CAATCTCTCAACATGATTGAACCATTTGGGATGGAGAAGCACCTTTGCTCTCAGCCACCT1508     GTTACTAAGTCAGGAGTGTAGTTGGATCTCTACATTAATGTCCTCTTGCTGTCTACAGTA1568     GCTGCTACCTAAAAAAAGATGTTTTATTTTGCCAGTTGGACACAGGTGATTGGCTCCTGG1628     GTTTCATGTTCTGTGACATCCTGCTTCTTCTTCCAAATGCAGTTCATTGCAGACACCACC1688     ATATTGCTATCTAATGGGGAAATGTAGCTATGGGCCATAACCAAAACTCACATGAAACGG1748     AGGCAGATGGAGACCAAGGGTGGGATCCAGAATGGAGTCTTTTCTGTTATTGTATTTAAA1808     AGGGTAATGTGGCCTTGGCATTTCTTCTTAGAAAAAAACTAATTTTTGGTGCTGATTGGC1868     ATGTCTGGTTCACAGTTTAGCATTGTTATAAACCATTCCATTCGAAAAGCACTTTGAAAA1928     ATTGTTCCCGAGCGATAGATGGGATGGTTTATGCAGGAATTC1970     (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 309 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     MetGluLeuLeuCysHisGluValAspProValArgArgAlaValArg     151015     AspArgAsnLeuLeuGlyAspAspArgValLeuGlnAsnLeuLeuThr     202530     IleGluPheProProGlyLeuAlaMetGluLeuLeuCysHisGluVal     354045     AspProValArgArgGluGluArgTyrLeuProGlnCysSerTyrPhe     505560     LysCysValGlnLysAspIleGlnProTyrMetArgArgMetValAla     65707580     ThrTrpMetLeuGluValCysGluGluGlnLysCysGluGluGluVal     859095     PheProLeuAlaMetAsnTyrLeuAspArgPheLeuAlaGlyValPro     100105110     ThrProLysSerHisLeuGlnLeuLeuGlyAlaValCysMetPheLeu     115120125     AlaSerLysLeuLysGluThrSerProLeuThrAlaGluLysLeuCys     130135140     IleTyrThrAspAsnSerIleLysProGlnGluLeuLeuGluTrpGlu     145150155160     LeuValValLeuGlyLysLeuLysTrpAsnLeuAlaAlaValThrPro     165170175     HisAspPheIleGluHisIleLeuArgLysLeuProGlnGlnArgGlu     180185190     LysLeuSerLeuIleArgLysHisAlaGlnThrPheIleAlaLeuCys     195200205     AlaThrAspPheLysPheAlaMetTyrProProSerMetIleAlaThr     210215220     GlySerValGlyAlaAlaIleCysGlyLeuGlnGlnAspGluGluVal     225230235240     SerSerLeuThrCysAspAlaLeuThrGluLeuLeuAlaLysIleThr     245250255     AsnThrAspValAspCysLeuLysAlaCysGlnAspGlnIleGluAla     260265270     ValLeuLeuAsnSerLeuGlnGlnTyrArgGlnAspGlnArgAspGly     275280285     SerLysSerGluAspGluLeuAspGlnAlaSerThrProThrAspVal     290295300     ArgAspIleAspLeu     305     (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1926 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 101..940     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     GAATTCCGATCCCCAGCCCGCCCGCCCGCGCTCTCCGGCCCGTCGCCTGCCTTGGGACTC60     GCGAGCCCGCACTCCCGCCCTGCCTGTTCGCTGCCCGAGTATGGAGCTGCTGTGT115     MetGluLeuLeuCys     15     TGCGAAGGCACCCGGCACGCGCCCCGGGCCGGGCCGGACCCGCGGCTG163     CysGluGlyThrArgHisAlaProArgAlaGlyProAspProArgLeu     101520     CTGGGGGACCAGCGTGTCCTGCAGAGCCTGCTCCGCCTGGAGGAGCGC211     LeuGlyAspGlnArgValLeuGlnSerLeuLeuArgLeuGluGluArg     253035     TACGTACCCCGCGCCTCCTACTTCCAGTGCGTGCAGCGGGAGATCAAG259     TyrValProArgAlaSerTyrPheGlnCysValGlnArgGluIleLys     404550     CCGCACATGCGGAAGATGCTGGCTTACTGGATGCTGGAGGTATGTGAG307     ProHisMetArgLysMetLeuAlaTyrTrpMetLeuGluValCysGlu     556065     GAGCAGCGCTGTGAGGAGGAAGTCTTCCCCCTGGCCATGAACTACCTG355     GluGlnArgCysGluGluGluValPheProLeuAlaMetAsnTyrLeu     70758085     GATCGCTACCTGTCTTGCGTCCCCACCCGAAAGGCGCAGTTGCAGCTC403     AspArgTyrLeuSerCysValProThrArgLysAlaGlnLeuGlnLeu     9095100     CTGGGTGCGGTCTGCATGGCCCCTGACCATCGAAAAACTGTGCATCTA451     LeuGlyAlaValCysMetAlaProAspHisArgLysThrValHisLeu     105110115     CACCGACCACGCTGTCGCCAGTTGCGGGACTGGGAGGTGCTGGTCCTA499     HisArgProArgCysArgGlnLeuArgAspTrpGluValLeuValLeu     120125130     GGGAAGCTCAAGTGGGACCTGGCTGCTGTGATTGCACATGATTTCCTG547     GlyLysLeuLysTrpAspLeuAlaAlaValIleAlaHisAspPheLeu     135140145     GCCTTCATTCTGCACCGGCTCTCTCTGCCCCGTGACCGACAGGCCTTG595     AlaPheIleLeuHisArgLeuSerLeuProArgAspArgGlnAlaLeu     150155160165     GTCAAAAAGCATGCCCAGACCTTTTTGGCCCTCTGTGCTACAGATTAT643     ValLysLysHisAlaGlnThrPheLeuAlaLeuCysAlaThrAspTyr     170175180     ACCTTTGCCATGTACCCGCCATCCATGATCGCCACGGGCAGCATTGGG691     ThrPheAlaMetTyrProProSerMetIleAlaThrGlySerIleGly     185190195     GCTGCAGTGCAAGGCCTGGGTGCCTGCTCCATGTCCGGGGATGAGCTC739     AlaAlaValGlnGlyLeuGlyAlaCysSerMetSerGlyAspGluLeu     200205210     ACAGAGCTGCTGGCAGGGATCACTGGCACTGAAGTGGACTGCCTGCGG787     ThrGluLeuLeuAlaGlyIleThrGlyThrGluValAspCysLeuArg     215220225     GCCTGTCAGGAGCAGATCGAAGCTGCACTCAGGGAGAGCCTCAGGGAA835     AlaCysGlnGluGlnIleGluAlaAlaLeuArgGluSerLeuArgGlu     230235240245     GCCGCTCAGACCAGCTCCAGCCCAGCGCCCAAAGCCCCCCGGGGCTCC883     AlaAlaGlnThrSerSerSerProAlaProLysAlaProArgGlySer     250255260     AGCAGCCAAGGGCCCAGCCAGACCAGCACTCTTACAGATGTCACAGCC931     SerSerGlnGlyProSerGlnThrSerThrLeuThrAspValThrAla     265270275     ATACACCTGTAGCCCTGGAGAGGCCCTCTGGAGTGGCCACTAAGCAGAG980     IleHisLeu     280     GAGGGGCCGCTGCACCCACCTCCCTGCCTCCAGGAACCACACCACATCTAAGCCTGAAGG1040     GGCGTCTGTTCCCCCTTCACAAAGCCCAAGGGATCTGGTCCTACCCATCCCCGCAGTGTG1100     CACTAAGGGGCCCGGCCAGCCATGTCTGCATTTCGGTGGCTAGTCAAGCTCCTCCTCCCT1160     GCATCTGACCAGCAGCGCCTTTCCCAACTCTAGCTGGGGGTGGGCCAGGCTGATGGGACA1220     GAATTGGATACATACACCAGCATTCCTTTTGAACGCCCCCCCCCACCCCTGGGGGCTCTC1280     ATGTTTTCAACTGCCAAAATGCTCTAGTGCCTTCTAAAGGTGTTGTCCCTTCTAGGGTTA1340     TTGCATTTGGATTGGGGTCCCTCTAAAATTTAATGCATGATAGACACATATGAGGGGGAA1400     TAGTCTAGATGGCTCCTCTCAGTACTTTGGAGGCCCCTATGTAGTCCTGGCTGACAGCTG1460     CTCCTAGAGGGAGGGGCCTAGGCTCAGCCAGAGAAGCTATAAATTCCTCTTTGCTTTGCT1520     TTCTGCTCAGCTTCTCCTGTGTGATTGACAGCTTTGCTGCTGAAGGCTCATTTTAATTTA1580     TTAATTGCTTTGAGCACAACTTTAAGAGGACGTAATGGGGTCCTGGCCATCCCACAAGTG1640     GTGGTAACCCTGGTGGTTGCTGTTTTCCTCCCTTCTGCTACTGGCAAAAGGATCTTTGTG1700     GCCAAGGAGCTGCTATAGCCTGGGGTGGGGTCATGCCCTCCTCTCCCATTGTCCCTCTGC1760     CCCATCCTCCAGCAGGGAAAATGCAGCAGGGATGCCCTGGAGGTGCTGAGCCCCTGTCTA1820     GAGAGGGAGGCAAGCCTGTTGACACAGGTCTTTCCTAAGGCTGCAAGGTTTAGGCTGGTG1880     GCCCAGGACCATCATCCTACTGTAATAAAGATGATTGTGGGAATTC1926     (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 280 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     MetGluLeuLeuCysCysGluGlyThrArgHisAlaProArgAlaGly     151015     ProAspProArgLeuLeuGlyAspGlnArgValLeuGlnSerLeuLeu     202530     ArgLeuGluGluArgTyrValProArgAlaSerTyrPheGlnCysVal     354045     GlnArgGluIleLysProHisMetArgLysMetLeuAlaTyrTrpMet     505560     LeuGluValCysGluGluGlnArgCysGluGluGluValPheProLeu     65707580     AlaMetAsnTyrLeuAspArgTyrLeuSerCysValProThrArgLys     859095     AlaGlnLeuGlnLeuLeuGlyAlaValCysMetAlaProAspHisArg     100105110     LysThrValHisLeuHisArgProArgCysArgGlnLeuArgAspTrp     115120125     GluValLeuValLeuGlyLysLeuLysTrpAspLeuAlaAlaValIle     130135140     AlaHisAspPheLeuAlaPheIleLeuHisArgLeuSerLeuProArg     145150155160     AspArgGlnAlaLeuValLysLysHisAlaGlnThrPheLeuAlaLeu     165170175     CysAlaThrAspTyrThrPheAlaMetTyrProProSerMetIleAla     180185190     ThrGlySerIleGlyAlaAlaValGlnGlyLeuGlyAlaCysSerMet     195200205     SerGlyAspGluLeuThrGluLeuLeuAlaGlyIleThrGlyThrGlu     210215220     ValAspCysLeuArgAlaCysGlnGluGlnIleGluAlaAlaLeuArg     225230235240     GluSerLeuArgGluAlaAlaGlnThrSerSerSerProAlaProLys     245250255     AlaProArgGlySerSerSerGlnGlyProSerGlnThrSerThrLeu     260265270     ThrAspValThrAlaIleHisLeu     275280     (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 819 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     GlnLeuCysCysGluValGluThrIleArgArgAlaTyrProAspAla     151015     AsnLeuLeuAsnAspArgValLeuArgAlaMetLeuLysAlaGluGlu     202530     ThrCysAlaProSerValSerTyrPheLysCysValGlnLysGluVal     354045     LeuProSerMetArgLysIleValAlaThrTrpMetLeuGluValCys     505560     GluGluGlnLysCysGluGluGluValPheProLeuAlaMetAsnTyr     65707580     LeuAspArgPheLeuSerLeuGluProValLysLysSerArgLeuGln     859095     LeuLeuGlyAlaThrCysMetPheSerIleValLeuGluAspGluLys     100105110     ProValSerValAsnGluValProAspTyrHisGluAspIleHisThr     115120125     TyrLeuArgGluMetGluValLysCysLysProLysValGlyTyrMet     130135140     LysLysGlnProAspIleThrAsnSerMetArgAlaIleLeuValAsp     145150155160     TrpLeuValGluValGlyGluGluTyrLysLeuGlnAsnGluThrLeu     165170175     HisLeuAlaValAsnTyrIleAspArgPheLeuSerSerMetSerVal     180185190     LeuArgGlyLysLeuGlnLeuValGlyThrAlaAlaMetLeuLysGlu     195200205     LeuProProArgAsnAspArgGlnArgPheLeuGluValValGlnTyr     210215220     GlnMetAspIleLeuGluTyrPheArgGluSerGluLysLysHisArg     225230235240     ProLysProArgTyrMetArgArgGlnLysAspIleSerHisAsnMet     245250255     ArgSerIleLeuIleAspTrpLeuValGluValSerGluGluTyrLys     260265270     LeuAspThrGluThrLeuTyrLeuSerValPheTyrLeuAspArgPhe     275280285     LeuSerGlnMetAlaValValArgSerLysLeuGlnLeuValGlyThr     290295300     AlaAlaMetTyrValAsnAspValAspAlaGluAspGlyAlaAspPro     305310315320     AsnLeuCysSerGluTyrValLysAspIleTyrAlaTyrLeuArgGln     325330335     LeuGluGluGluGlnAlaValArgProLysTyrLeuLeuGlyArgGlu     340345350     ValThrGlyAsnMetArgAlaIleLeuIleAspTrpLeuValGlnVal     355360365     GlnMetLysPheArgLeuLeuGlnGluThrMetTyrMetThrValSer     370375380     IleIleAspArgPheMetGlnAsnAsnCysValProLysLysMetLeu     385390395400     GlnLeuValGlyValThrAlaMetPheTrpAspAspLeuAspAlaGlu     405410415     AspTrpAlaAspProLeuMetValSerGluTyrValValAspIlePhe     420425430     GluTyrLeuAsnGluLeuGluIleGluThrMetProSerProThrTyr     435440445     MetAspArgGlnLysGluLeuAlaTrpLysMetArgGlyIleLeuThr     450455460     AspTrpLeuIleGluValHisSerArgPheArgLeuLeuProGluThr     465470475480     LeuPheLeuAlaValAsnIleIleAspArgPheLeuSerLeuArgVal     485490495     CysSerLeuAsnLysLeuGlnLeuValGlyIleAlaAlaLeuPheIle     500505510     GluLeuSerAsnAlaGluLeuLeuThrHisTyrGluThrIleGlnGlu     515520525     TyrHisGluGluIleSerGlnAsnValLeuValGlnSerSerLysThr     530535540     LysProAspIleLysLeuIleAspGlnGlnProGluMetAsnProHis     545550555560     GlnThrArgGluAlaIleValThrPheLeuTyrGlnLeuSerValMet     565570575     ThrArgValSerAsnGlyIlePhePheHisSerValArgPheTyrAsp     580585590     ArgTyrCysSerLysArgValValLeuLysAspGlnAlaLysLeuVal     595600605     ValGlyThrCysLeuTrpProAsnLeuValLysArgGluLeuGlnAla     610615620     HisHisSerAlaIleSerGluTyrAsnAsnAspGlnLeuAspHisTyr     625630635640     PheArgLeuSerHisThrGluArgProLeuTyrAsnLeuAsnSerGln     645650655     ProGlnValAsnProLysMetArgPheLeuIlePheAspPheIleMet     660665670     TyrCysHisThrArgLeuAsnLeuSerThrSerThrLeuPheLeuThr     675680685     PheThrIleLeuAspLysTyrSerSerArgPheIleIleLysSerTyr     690695700     AsnTyrGlnLeuLeuSerLeuThrAlaLeuTrpValAlaSerLysMet     705710715720     LysGluThrIleProLeuThrAlaGluLysLeuCysIleTyrThrAsp     725730735     GlySerIleArgProGluGluLeuLeuGlnMetGluLeuLeuLeuVal     740745750     AsnLysLeuLysTrpAsnLeuAlaAlaMetThrProHisGluPheIle     755760765     GluHisPheLeuSerLysMetProGluAlaGluGluAsnLysGlnIle     770775780     IleArgLysHisAlaGlnThrPheValAlaLeuCysAlaThrAspVal     785790795800     LysPheIleSerAsnProProSerMetValAlaAlaGlySerValVal     805810815     AlaAlaVal     (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 100 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     LeuAlaSerLysPheGluGluIleTyrProProGluValAlaGluPhe     151015     ValTyrIleThrValAspThrTyrThrLysLysGlnValLeuArgMet     202530     GluHisLeuValLeuLysValLeuThrPheAspLeuAlaAlaProThr     354045     ValAsnGlnPheLeuThrGlnTyrPheLeuHisGlnGlnAsnCysLys     505560     ValGluSerLeuAlaMetPheLeuGlyGluLeuSerLeuIleAspAla     65707580     AspProTyrLeuLysTyrLeuProSerValIleAlaGlyAlaAlaPhe     859095     HisLeuAlaLeu     100     (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 101 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     IleAlaAlaLysTyrGluGluIleTyrProProGluValGlyGluPhe     151015     ValPheLeuThrAspAspSerTyrThrLysAlaGlnValLeuArgMet     202530     GluGlnValIleLeuLysIleLeuSerPheAspLeuCysThrProThr     354045     AlaTyrValPheIleAsnThrTyrAlaValLeuCysAspMetProGlu     505560     LysLeuLysTyrMetThrLeuTyrIleSerGluLeuSerLeuMetGlu     65707580     GlyGluThrTyrLeuGlnTyrLeuProSerLeuMetSerSerAlaSer     859095     ValAlaLeuAlaArg     100     (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 100 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     IleAlaSerLysTyrGluGluMetTyrProProGluIleGlyAspPhe     151015     AlaPheValThrAspAsnThrTyrThrLysHisGlnIleArgGlnMet     202530     GluMetLysIleLeuArgAlaLeuAsnPheGlyLeuGlyArgProLeu     354045     ProLeuHisPheLeuArgArgAlaSerLysIleGlyGluValAspVal     505560     GluGlnHisThrLeuAlaLysTyrLeuMetGluLeuThrMetLeuAsp     65707580     TyrAspMetValHisPheProProSerGlnIleAlaAlaGlyAlaPhe     859095     CysLeuAlaLeu     100     (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 100 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     IleAlaSerLysTyrGluGluValMetCysProSerValGlnAsnPhe     151015     ValTyrMetAlaAspGlyGlyTyrAspGluGluGluIleLeuGlnAla     202530     GluArgTyrIleLeuArgValLeuGluPheAsnLeuAlaTyrProAsn     354045     ProMetAsnPheLeuArgArgIleSerLysAlaAspPheTyrAspIle     505560     GlnThrArgThrValAlaLysTyrLeuValGluIleGlyLeuLeuAsp     65707580     HisLysLeuLeuProTyrProProSerGlnGlnCysAlaAlaAlaMet     859095     TyrLeuAlaArg     100     (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 51 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     LeuAlaAlaLysThrTrpGlyArgLeuSerGluLeuValHisTyrCys     151015     GlyGlySerAspLeuPheAspGluSerMetPheIleGlnMetGluArg     202530     HisIleLeuAspThrLeuAsnTrpAspValTyrGluProMetIleAsn     354045     AspTyrIle     50     (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 51 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     IleSerSerLysPheTrpAspArgMetAlaThrLeuLysValLeuGln     151015     AsnLeuCysCysAsnGlnTyrSerIleLysGlnPheThrThrMetGlu     202530     MetHisLeuPheLysSerLeuAspTrpSerIleSerAlaThrPheAsp     354045     SerTyrIle     50     (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 16 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     CCCAAAAACTGTCTTT16     (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 31 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     CCCAAAAACTGTCTTTAAAAGAGAGAGAGAG31     (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     TGCATAACCCTGAGCGGTGGGGGAGGAGGGTT32     (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     TGCATAACCCTGAGCGGTGGGGGAGGAGGGTT32     (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 32 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     TGCATAACCCTGAGCGGTGGGGGAGGAGGGTT32     (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 295 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     MetGluHisGlnLeuLeuCysCysGluValGluThrIleArgArgAla     151015     TyrProAspAlaAsnLeuLeuAsnAspArgValLeuArgAlaMetLeu     202530     LysAlaGluGluThrCysAlaProSerValSerTyrPheLysCysVal     354045     GlnLysGluValLeuProSerMetArgLysIleValAlaThrTrpMet     505560     LeuGluValCysGluGluGlnLysCysGluGluGluValPheProLeu     65707580     AlaMetAsnTyrLeuAspArgPheLeuSerLeuGluProValLysLys     859095     SerArgLeuGlnLeuLeuGlyAlaThrCysMetPheValAlaSerLys     100105110     MetLysGluThrIleProLeuThrAlaGluLysLeuCysIleTyrThr     115120125     AspGlySerIleArgProGluGluLeuLeuGlnMetGluLeuLeuLeu     130135140     ValAsnLysLeuLysTrpAsnLeuAlaAlaMetThrProHisAspPhe     145150155160     IleGluHisPheLeuSerLysMetProGluAlaGluGluAsnLysGln     165170175     IleIleArgLysHisAlaGlnThrPheValAlaLeuCysAlaThrAsp     180185190     ValLysPheIleSerAsnProProSerMetValAlaAlaGlySerVal     195200205     ValAlaAlaValLysGlyLeuAsnLeuArgSerProAsnAsnPheLeu     210215220     SerTyrTyrArgLeuThrArgPheLeuSerArgValIleLysCysAsp     225230235240     ProAspCysLeuArgAlaCysGlnGluGlnIleGluAlaLeuLeuGlu     245250255     SerSerLeuArgGlnAlaGlnGlnAsnMetAspProLysAlaAlaGlu     260265270     GluGluGluGluGluGluGluGluValAspLeuAlaCysThrProThr     275280285     AspValArgAspValAspIle     290295     (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 295 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     MetGluAsnGlnLeuLeuCysCysGluValGluThrIleArgArgAla     151015     TyrProAspThrAsnLeuLeuAsnAspArgValLeuArgAlaMetLeu     202530     LysThrGluGluThrCysAlaProSerValSerTyrPheLysCysVal     354045     GlnLysGluIleValProSerMetArgLysIleValAlaThrTrpMet     505560     LeuGluValCysGluGluGlnLysCysGluGluGluValPheProLeu     65707580     AlaMetAsnTyrLeuAspArgPheLeuSerLeuGluProLeuLysLys     859095     SerArgLeuGlnLeuLeuGlyAlaThrCysMetPheValAlaSerLys     100105110     MetLysGluThrIleProLeuThrAlaGluLysLeuCysIleTyrThr     115120125     AspAsnSerIleArgProGluGluLeuLeuGlnMetGluLeuLeuLeu     130135140     ValAsnLysLeuLysTrpAsnLeuAlaAlaMetThrProHisAspPhe     145150155160     IleGluHisPheLeuSerLysMetProAspAlaGluGluAsnLysGln     165170175     IleIleArgLysHisAlaGlnThrPheValAlaLeuCysAlaThrAsp     180185190     ValLysPheIleSerAsnProProSerMetValAlaAlaGlySerMet     195200205     ValAlaAlaMetGlnGlyLeuAsnLeuGlySerProAsnAsnPheLeu     210215220     SerArgTyrArgThrThrHisPheLeuSerArgValIleLysCysAsp     225230235240     ProAspCysLeuArgAlaCysGlnGluGlnIleGluAlaLeuLeuGlu     245250255     SerSerLeuArgGlnAlaGlnGlnAsnMetAspProLysAlaThrGlu     260265270     GluGluGlyGluValGluGluGluAlaGlyLeuAlaCysThrProThr     275280285     AspValArgAspValAspIle     290295     (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 189 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     MetGluLeuLeuCysHisGluValAspProValArgArgAlaValArg     151015     AspArgAsnLeuLeuArgAspAspArgValLeuGlnAsnLeuLeuThr     202530     IleGluGluArgTyrLeuProGlnCysSerTyrPheLysCysValGln     354045     LysAspIleGlnProTyrMetArgArgMetValAlaThrTrpMetLeu     505560     GluValCysGluGluGlnLysCysGluGluGluValPheProLeuAla     65707580     MetAsnTyrLeuAspArgPheLeuAlaGlyValProThrProLysSer     859095     HisProProSerMetIleAlaThrGlySerValGlyAlaAlaIleCys     100105110     GlyLeuLysGlnAspGluGluValSerSerLeuThrCysAspAlaLeu     115120125     ThrGluLeuLeuAlaLysIleThrAsnThrAspValAspCysLeuLys     130135140     AlaCysGlnGluGlnIleGluAlaValLeuLeuAsnSerLeuGlnGln     145150155160     TyrArgGlnAspGlnArgAspGlySerLysSerGluAspGluLeuAsp     165170175     GlnAlaSerThrProThrAspValArgAspIleAspLeu     180185     (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 236 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     MetArgArgMetValAlaThrTrpMetLeuGluValCysGluGluGln     151015     LysCysGluGluGluValPheProLeuAlaMetAsnTyrLeuAspArg     202530     PheLeuAlaGlyValProThrProLysThrHisLeuGlnLeuLeuGly     354045     AlaValCysMetPheLeuAlaSerLysLeuLysGluThrIleProLeu     505560     ThrAlaGluLysLeuCysIleTyrThrAspAsnSerValLysProGln     65707580     GluLeuLeuGluTrpGluLeuValValLeuGlyLysLeuLysTrpAsn     859095     LeuAlaAlaValThrProHisAspPheIleGluHisIleLeuArgLys     100105110     LeuProGlnGlnLysGluLysLeuSerLeuIleArgLysHisAlaGln     115120125     ThrPheIleAlaLeuCysAlaThrAspPheLysPheAlaMetTyrPro     130135140     ProSerMetIleAlaThrGlySerValGlyAlaAlaIleCysGlyLeu     145150155160     GlnGlnAspAspGluValAsnThrLeuThrCysAspAlaLeuThrGlu     165170175     LeuLeuAlaLysIleThrHisThrAspValAspCysLeuLysAlaCys     180185190     GlnGluGlnIleGluAlaLeuLeuLeuAsnSerLeuGlnGlnPheArg     195200205     GlnGluGlnHisAsnAlaGlySerLysSerValGluAspProAspGln     210215220     AlaThrThrProThrAspValArgAspValAspLeu     225230235     (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 292 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     MetGluLeuLeuCysCysGluGlyThrArgHisAlaProArgAlaGly     151015     ProAspProArgLeuLeuGlyAspGlnArgValLeuGlnSerLeuLeu     202530     ArgLeuGluGluArgTyrValProArgAlaSerTyrPheGlnCysVal     354045     GlnArgGluIleLysProHisMetArgLysMetLeuAlaTyrTrpMet     505560     LeuGluValCysGluGluGlnArgCysGluGluGluValPheProLeu     65707580     AlaMetAsnTyrLeuAspArgTyrLeuSerCysValProThrArgLys     859095     AlaGlnLeuGlnLeuLeuGlyAlaValCysMetLeuLeuAlaSerLys     100105110     LeuArgGluThrThrProLeuThrIleGluLysLeuCysIleTyrThr     115120125     AspHisAlaValSerProArgGlnLeuArgAspTrpGluValLeuVal     130135140     LeuGlyLysLeuLysTrpAspLeuAlaAlaValIleAlaHisAspPhe     145150155160     LeuAlaPheIleLeuHisArgLeuSerLeuProArgAspArgGlnAla     165170175     LeuValLysLysHisAlaGlnThrPheLeuAlaLeuCysAlaThrAsp     180185190     TyrThrPheAlaMetTyrProProSerMetIleAlaThrGlySerIle     195200205     GlyAlaAlaValGlnGlyLeuGlyAlaCysSerMetSerGlyAspGlu     210215220     LeuThrGluLeuLeuAlaGlyIleThrGlyThrGluValAspCysLeu     225230235240     ArgAlaCysGlnGluGlnIleGluAlaAlaLeuArgGluSerLeuArg     245250255     GluAlaAlaGlnThrSerSerSerProAlaProLysAlaProArgGly     260265270     SerSerSerGlnGlyProSerGlnThrSerThrProThrAspValThr     275280285     AlaIleHisLeu     290     (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 237 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     MetArgLysMetLeuAlaTyrTrpMetLeuGluValCysGluGluGln     151015     ArgCysGluGluAspValPheProLeuAlaMetAsnTyrLeuAspArg     202530     TyrLeuSerCysValProThrArgLysAlaGlnLeuGlnLeuLeuGly     354045     ThrValCysIleLeuLeuAlaSerLysLeuArgGluThrThrProLeu     505560     ThrIleGluLysLeuCysIleTyrThrAspGlnAlaValAlaProTrp     65707580     GlnLeuArgGluTrpGluValLeuValLeuGlyLysLeuLysTrpAsp     859095     LeuAlaAlaValIleAlaHisAspPheLeuAlaLeuIleLeuHisArg     100105110     LeuSerLeuProSerAspArgGlnAlaLeuValLysLysHisAlaGln     115120125     ThrPheLeuAlaLeuCysAlaThrAspTyrThrPheAlaMetTyrPro     130135140     ProSerMetIleAlaThrGlySerIleGlyAlaAlaValIleGlyLeu     145150155160     GlyAlaCysSerMetSerAlaAspGluLeuThrGluLeuLeuAlaGly     165170175     IleThrGlyThrGluValAspCysLeuArgAlaCysGlnGluGlnIle     180185190     GluAlaAlaLeuArgGluSerLeuArgGluAlaAlaGlnThrAlaPro     195200205     SerProValProLysAlaProArgGlySerSerSerGlnGlyProSer     210215220     GlnThrSerThrProThrAspValThrAlaIleHisLeu     225230235     (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 106 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     MetArgAlaIleLeuValAspTrpLeuValGluValGlyGluGluTyr     151015     LysLeuGlnAsnGluThrLeuHisLeuAlaValAsnTyrIleAspArg     202530     PheLeuSerSerMetSerValLeuArgGlyLysLeuGlnLeuValGly     354045     ThrAlaAlaMetLeuLeuAlaSerLysPheGluGluIleTyrProPro     505560     GluValAlaGluPheValTyrIleThrAspAspThrTyrThrLysLys     65707580     GlnValLeuArgMetGluHisLeuValLeuLysValLeuThrPheAsp     859095     LeuAlaAlaProThrValAsnGlnPheLeu     100105     (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 106 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     MetArgAlaIleLeuIleAspTrpLeuValGlnValGlnMetLysPhe     151015     ArgLeuLeuGlnGluThrMetTyrMetThrValSerIleIleAspArg     202530     PheMetGlnAsnAsnCysValProLysLysMetLeuGlnLeuValGly     354045     ValThrAlaMetPheIleAlaSerLysTyrGluGluMetTyrProPro     505560     GluIleGlyAspPheAlaPheValThrAspAsnThrTyrThrLysHis     65707580     GlnIleArgGlnMetGluMetLysIleLeuArgAlaLeuAsnPheGly     859095     LeuGlyArgProLeuProLeuHisPheLeu     100105     (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 106 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     MetArgAlaIleLeuValAspTrpLeuValGlnValHisSerLysPhe     151015     ArgLeuLeuGlnGluThrLeuTyrMetCysValGlyIleMetAspArg     202530     PheLeuGlnValGlnProValSerArgLysLysLeuGlnLeuValGly     354045     IleThrAlaLeuLeuLeuAlaSerLysTyrGluGluMetPheSerPro     505560     AsnIleGluAspPheValTyrIleThrAspAsnAlaTyrThrSerSer     65707580     GlnIleArgGluMetGluThrLeuIleLeuLysGluLeuLysPheGlu     859095     LeuGlyArgProLeuProLeuHisPheLeu     100105     (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 105 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     LeuGlnIlePhePheThrAsnValIleGlnAlaLeuGlyGluHisLeu     151015     LysLeuArgGlnGlnValIleAlaThrAlaThrValTyrPheLysArg     202530     PheTyrAlaArgTyrSerLeuLysSerIleAspProValLeuMetAla     354045     ProThrCysValPheLeuAlaSerLysValGluGluIleLeuLysThr     505560     ArgPheSerTyrAlaPheProLysGluPheProTyrArgMetAsnHis     65707580     IleLeuGluCysGluPheTyrLeuLeuGluLeuMetAspCysCysLeu     859095     IleValTyrHisProTyrArgProLeu     100105     (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 105 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     MetArgAlaIleLeuLeuAspTrpLeuMetGluValCysGluValTyr     151015     LysLeuHisArgGluThrPheTyrLeuAlaGlnAspPhePheAspArg     202530     TyrMetAlaGluAsnValValLysThrLeuLeuGlnLeuIleGlyIle     354045     SerSerLeuPheIleAlaAlaLysLeuGluGluIleTyrProProLys     505560     LeuHisGlnPheAlaTyrValThrAspGlyAlaCysSerGlyAspGlu     65707580     IleLeuThrMetGluLeuMetIleMetLysAlaLeuLysTrpArgLeu     859095     SerProLeuThrIleValSerTrpLeu     100105     (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1462 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: join(378..569, 662..1000, 1040..1189,     1191..1292, 1292..1324)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     TGATCAAGTTGACACTCAATATTAACCCTCATAGACTGTGATCCCTATGTTGCTGCCTTC60     CCTCGTTTCTATTGCTCTTTGGCCCCAACCCAAATAAGGTTCCTTGGGACACACTAAAGA120     AGGAGGTGGAGTTCGAAGGGGAGGAGAGATGTGAGCGAGGCAGGCAGGGAAGCTCTGCTC180     GCCCACTGCCCAATCCTCACCTCTCTTCTCCTCCACCTTCTGTCTCTGCCCTCACCTCTC240     CTCTGAAAACCCCCTATTGAGCCAAAGGAAGGAGATGAGGGGAATGCTTTTGCCTTCCCC300     CTCCAAAACAAAAACAAAAACAAACACACTTTTCCAGTCCAGAGAAAGCAGGGGAGTGAG360     GGGTCACAGAGCTGGCCATGCAGCTGCTGGGCTGTGAGGTAGACCCGGTC410     MetGlnLeuLeuGlyCysGluValAspProVal     1510     CTCAGAGCCACGAGGGACTGCAACCTACTCCAAGTTGACCGTGTCCTG458     LeuArgAlaThrArgAspCysAsnLeuLeuGlnValAspArgValLeu     152025     AAGAACCTGCTTGCTATCAAGAAGCGCTACCTTCAGTAATGCTCCTAC506     LysAsnLeuLeuAlaIleLysLysArgTyrLeuGlnCysSerTyr     303540     TTCAAGTGTGTGCAGAAGGCCATCCAGCCGTACATGCACAGGATGGTG554     PheLysCysValGlnLysAlaIleGlnProTyrMetHisArgMetVal     455055     CCACTTCTGATGGTGGCCATTTGATTGGTGCCACTTCTGATGGTGGCCAACATGA609     ProLeuLeuMetVal     60     TTGAACCATTTGGGATGGAAAAGCACCTTTACTCTCAGCCACCTGTTAACTAATG664     Met     65     CTGGAGGTCTGTGAGGAACAGAAGTGTGAAGAAAAGGTTTTCCCTCTG712     LeuGluValCysGluGluGlnLysCysGluGluLysValPheProLeu     707580     GCCACGATTTACCTGGACTGTTTCTTCGCCAGGATCCCAACTTCAAAG760     AlaThrIleTyrLeuAspCysPhePheAlaArgIleProThrSerLys     859095     TCCCATCTGCAACTCCTGGGTGCTGTCTGCATGTTCCTGGCCTCCAGG808     SerHisLeuGlnLeuLeuGlyAlaValCysMetPheLeuAlaSerArg     100105110     CTCAAAGAGTCCAGCCCACTGACTGCCAAAAAGCTGTGCATTTATACC856     LeuLysGluSerSerProLeuThrAlaLysLysLeuCysIleTyrThr     115120125     GACAACTCCATCAAGCCTCAGGAGCTGCTGGAGTGGGAACTGGTGGTG904     AspAsnSerIleLysProGlnGluLeuLeuGluTrpGluLeuValVal     130135140145     TTGGGAAAGTTGAAGTGGAACCTGGCAGCTGTCACGCCTCATGACTTC952     LeuGlyLysLeuLysTrpAsnLeuAlaAlaValThrProHisAspPhe     150155160     ATTTAGTACATCTTGCACAAGCTGCCCCAGCAGCGGGAGAAGCTGTCT1000     IleTyrIleLeuHisLysLeuProGlnGlnArgGluLysLeuSer     165170175     CCAATCTGCAAGCAAGTCCAGAACTTCAATGCTCTGTATGCAATGTACCCGCCA1054     AlaMetTyrProPro     180     TCAATGGTTGCAACTGGAAGTGTAGGAGCAGCTATCTGTGGACTTCAG1102     SerMetValAlaThrGlySerValGlyAlaAlaIleCysGlyLeuGln     185190195     CAACATGAGGAAGTGAGCTCACTCCCTTGCAATGCCCTGACTGAGCTG1150     GlnHisGluGluValSerSerLeuProCysAsnAlaLeuThrGluLeu     200205210     CTGGCAAAGATCACCAACACAGATGTGGATTGTCTCAAAAGCCAAC1196     LeuAlaLysIleThrAsnThrAspValAspCysLeuLysAlaAsn     215220225     CGGGAGCATATTGAGGTGGTCTTCCTCAACAGCCTGCAGCAGTGCCAT1244     ArgGluHisIleGluValValPheLeuAsnSerLeuGlnGlnCysHis     230235240245     CAGGACCAGCAGGACAGATCCAAGTCAGAGGATGAACTGGGCCAAGCA1292     GlnAspGlnGlnAspArgSerLysSerGluAspGluLeuGlyGlnAla     250255260     AGCACCCCTATAGACCTGTGAGATATCGACCTGGAGGATGGCAGTCCAGCTGA1345     SerThrProIleAspLeuAspIleAspLeu     265270     GAGGCGCATTCATAATCTGCTGTCTCCTTCTTTCTGGTTATGTTTTGTTCTTTGTATCTT1405     AGGGCGAAACTTAAAAAAAAAAACCTCTGCCCCCACATAGTTCGTGTTTAAAGATCT1462     (2) INFORMATION FOR SEQ ID NO:31:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 39 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: C-terminal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     MetGlnLeuLeuGlyCysGluValAspProValLeuArgAlaThrArg     151015     AspCysAsnLeuLeuGlnValAspArgValLeuLysAsnLeuLeuAla     202530     IleLysLysArgTyrLeuGln     35     (2) INFORMATION FOR SEQ ID NO:32:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2022 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: join(1137..1211, 1211..1678, 1680..1790)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     AAGCTTCCAGATTAGAAAAGAAAAAATAAAACTATCTTTATTTGCAGATGACATGATCGG60     TCCATTCTCATGCTGCTTATAAAGACATACCCAAGACTGGATAATTTATAAAGGAAAGAG120     GTTTGGCTCACAGTTCCCCATGGGTGGAGAGGCCTCACAATCATGGCGAAAGAGCAAGGA180     GCATCTCACATGGCAGCAGGCAAGAAAAGAATGAGAGCCACGCCAGAGGGAAACCCCTTA240     TAAAATCATCAGATCTCGAGAGACTTATTCACTGTCAGGAGAACAGTATGGAGGAAACGC300     CCTTATGATTCAATTATCTCGCACTGTGTTCCTCCCACAACACATGGGAATTATGGGAGC360     TACAATTCAAGATGAGATTTGGGTGGAGACACAGCCAAACCATATCAATCTTTTTTTTCT420     TATTCTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCCCACTCTGTTATCTAGGCTGG480     AGTGCAGTGGTGTGTGATCTTGGCTCACTGCAACCTCAGCCTCCCAGGTTCAAGCGATTC540     TCCTGCCTCAGACTCCTGAATAGCTGAAATTACAGGCACCTGCCACTACGCCTGGCAAAT600     ATTTTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTTGAGACAGAGTCTCTCTCTGTCGCC660     CAGGCTGGAGTGCAGTGGGCGCGATCTCAGCTCACTGCAAACTCTGCTCCCGGGTTCAAG720     CCATTCTCCTGCCTCAGCTCCCAAGTAGCTGGGACTACAGGCGCCCACCACCACCATGCC780     AGGCTAATTTTTTGTATTTTTAGTAGAGACAGGGTTTCACCGTGTTAGCCAGGATGGTCT840     CAATCTCCTGACCTCGTGATCCGCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCG900     TGAGCCACTATGCCCAACCGTATCAATCTTGTATATAGAAAAACCTAAGGAATCTACAAA960     AAAACCCTATTATAACTAATATAATAATAATCTGCAAAGTTGTAGACTATGAGATCAATA1020     TACAAAAATTAACTCAATTTCTTTACATGTACAATGAATAACCCCAAAACAAAACTGGGA1080     ATATAATTCTATTTTTAATAGTATCACAAAGAATGACAATACTTAGAAACAAATGA1136     TGGGCGCTAGCTTGCACTCCCGCCCTGCCTGTGCGCTGCCCGAGTGTG1184     TrpAlaLeuAlaCysThrProAlaLeuProValArgCysProSerVal     151015     GAGCTGCTATGCTGCGAAGGCTCGAGGGACCCGCAGACGCCAGGGGAT1232     GluLeuLeuCysCysGluGlySerArgAspProGlnThrProGlyAsp     202530     CAGCGCGTCCTGCAGAGCTTGCTCCCCTTGGAGTAGCGCTGCGTGCAC1280     GlnArgValLeuGlnSerLeuLeuProLeuGluArgCysValHis     354045     TGCGCCTACTTCCAGTGCGTGCAAAGGGAGAGCAAGCCGCACATGCGG1328     CysAlaTyrPheGlnCysValGlnArgGluSerLysProHisMetArg     505560     AAGATGCTGGTTTACTGGATGCTGGAGGTGTGTGAGGAGCAGTGCTGT1376     LysMetLeuValTyrTrpMetLeuGluValCysGluGluGlnCysCys     65707580     GAGGAGGAGCAGTGCTGTAAGGAGGAAGTCTTTCCCCTGGCCATGAAC1424     GluGluGluGlnCysCysLysGluGluValPheProLeuAlaMetAsn     859095     CACCTGCATGCTACCTGTCCTACGTCCCCACCCACCCGAAAGGCACAG1472     HisLeuHisAlaThrCysProThrSerProProThrArgLysAlaGln     100105110     TTGCAGCTCTTGGTTGCGGTCTCCATGCGGCTGGCCTCCAAGCTGCGT1520     LeuGlnLeuLeuValAlaValSerMetArgLeuAlaSerLysLeuArg     115120125     AAGACTGGGCCCATGACCATTGAGAAAATGTGCATCTACACCGACCAC1568     LysThrGlyProMetThrIleGluLysMetCysIleTyrThrAspHis     130135140     GCTGTCTCTCCCTGCCAGTTGCGGGACTGGGAGGTGATGGTCCTGGGG1616     AlaValSerProCysGlnLeuArgAspTrpGluValMetValLeuGly     145150155160     AAGCTCAAATGGGACCTGGCCGCTGTGATTGCTCATGACTTCTTGGCC1664     LysLeuLysTrpAspLeuAlaAlaValIleAlaHisAspPheLeuAla     165170175     CTCATTCTGCACCGACCGACAGGCCTTGGTCAAAAAGCATGCCCAG1710     LeuIleLeuHisArgArgGlnAlaLeuValLysLysHisAlaGln     180185190     ATCTTTTTGGCTGTCTGTGCTACAGATTACACCTTTGCCATGTACCCA1758     IlePheLeuAlaValCysAlaThrAspTyrThrPheAlaMetTyrPro     195200205     CCATCCAGTTGTGAAAACAACCCAAATGCCTGTAACTGATGAACAGATAACCA1811     ProSerSerCysGluAsnAsnProAsnAlaCys     210215     TATGTGATATATATCAATACAATGGAATATGGCCTGGCATGCTGGCTTACGCTGTAATCC1871     TGCACTTTGGGAGGCCAAAGTGGAGGATCACTTGAGCCGAGGAGTTCAAGGCCAGCCTGG1931     GCACAAAGTGAGACTCCTTCTAAAAAAATAAAATAAAATAAAAAATAAAAACAATGTAAT1991     ATTATTCAGCCATAGAAAGGAATAAAGTACT2022     (2) INFORMATION FOR SEQ ID NO:33:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 43 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: C-terminal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     TrpAlaLeuAlaCysThrProAlaLeuProValArgCysProSerVal     151015     GluLeuLeuCysCysGluGlySerArgAspProGlnThrProGlyAsp     202530     GlnArgValLeuGlnSerLeuLeuProLeuGlu     3540     (2) INFORMATION FOR SEQ ID NO:34:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1317 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     GAGCTCGATCAGTACACTCGTTTGTTTAATTGATAATTGTCCTGAATTATGCCGGCTCCT60     GCAGCCCCCTCACGCTCACGAATTCAGTCCCAGGGCAAATTCTAAAGGTGAAGGGACGTC120     TACACCCCCAACAAAACCAATTAGGAACCTTCGGTGGGTCTTGTCCCAGGCAGAGGGGAC180     TAATATTTCCAGCAATTTAATTTCTTTTTTAATTAAAAAAAATGAGTCAGAATGGAGATC240     ACTGTTTCTCAGCTTTCCATTCAGAGGTGTGTTTCTCCCGGTTAAATTGCCGGCACGGGA300     AGGGAGGGGGTGCAGTTGGGGACCCCCGCAAGGACCGACTGGTCAAGGTAGGAAGGCAGC360     CCGAAGAGTCTCCAGGCTAGAAGGACAAGATGAAGGAAATGCTGGCCACCATCTTGGGCT420     GCTGCTGGAATTTTCGGGCATTTATTTTATTTTATTTTTTGAGCGAGCGCATGCTAAGCT480     GAAATCCCTTTAACTTTTAGGTTACCCCTTGGGCATTTGCAACGACGCCCCTGTGCGCCG540     GAATGAAACTTGCACAGGGGTTGTGTGCCCGGTCCTCCCCGTCCTTGCATGCTAAATTAG600     TTCTTGCAATTTACACGTGTTAATGAAAATGAAAGAAGATGCAGTCGCTGAGATTCTTTG660     GCCGTCTGTCCGCCCGTGGGTGCCCTCGTGGCGTTCTTGGAAATGCGCCCATTCTGCCGG720     CTTGGATATGGGGTGTCGCCGCGCCCCAGTCACCCCTTCTCGTGGTCTCCCCAGGCTGCG780     TGCTGGCCGGCCTTCCTAGTTGTCCCCTACTGCAGAGCCACCTCCACCTCACCCCCTAAA840     TCCCGGGACCCACTCGAGGCGGACGGGCCCCCTGCACCCCTCTCGGCGGGGAGAAAGGCT900     GCAGCGGGGCGATTTGCATTTCTATGAAAACCGGACTACAGGGGCAACTGCCCGCAGGGC960     AGCGCGGCGCCTCAGGGATGGCTTTTCGTCTGCCCCTCGCTGCTCCCGGCGTTCTGCCCG1020     CGCCCCCTCCCCCTGCGCCCGCCCCCGCCCCCCTCCCGCTCCCATTCTCTGCCGGGCTTT1080     GATCTTTGCTTAACAACAGTAACGTCACACGGACTACAGGGGAGTTTTGTTGAAGTTGCA1140     AAGTCCTGGAGCCTCCAGAGGGCTGTCGGCGCAGTAGCAGCGAGCAGCAGAGTCCGCACG1200     CTCCGGCGAGGGGCAGAAGAGCGCGAGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCG1260     CGGACCCAGCCAGGACCCACAGCCCTCCCCAGCTGCCCAGGAAGAGCCCCAGCCATG1317     (2) INFORMATION FOR SEQ ID NO:35:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1624 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     GAGCTCGAGCCACGCCATGCCCGCTGCACGTGCCAGCTTGGCCAGCACATCAGGGCGCTG60     GTCTCTCCCCTTCCTCCTGGAGTGAAATACACCAAAGGGCGCGGTGGGGGTGGGGGGTGA120     CGGGAGGAAGGAGGTGAAGAAACGCCACCAGATCGTATCTCCTGTAAAGACAGCCTTGAC180     TCAAGGATGCGTTAGAGCACGTGTCAGGGCCGACCGTGCTGGCGGCGACTTCACCGCAGT240     CGGCTCCCAGGGAGAAAGCCTGGCGAGTGAGGCGCGAAACCGGAGGGGTCGGCGAGGATG300     CGGGCGAAGGACCGAGCGTGGAGGCCTCATGCTCCGGGGAAAGGAAGGGGTGGTGGTGTT360     TGCGCAGGGGGAGCGAGGGGGAGCCGGACCTAATCCCTTCACTCGCCCCCTTCCCTCCCG420     GGCCATTTCCTAGAAAGCTGCATCGGTGTGGCCACGCTCAGCGCAGACACCTCGGGCGGC480     TTGTCAGCAGATGCAGGGGCGAGGAAGCGGGTTTTTCCTGCGTGGCCGCTGGCGCGGGGG540     AACCGCTGGGAGCCCTGCCCCCGGCCTGCGGCGGCCCTAGACGCTGCACCGCGTCGCCCC600     ACGGCGCCCGAAGAGCCCCCAGAAACACGATGGTTTCTGCTCGAGGATCACATTCTATCC660     CTCCAGAGAAGCACCCCCCTTCCTTCCTAATACCCACCTCTCCCTCCCTCTTCTTCCTCT720     GCACACACTCTGCAGGGGGGGGCAGAAGGGACGTTGTTCTGGTCCCTTTAATCGGGGCTT780     TCGAAACAGCTTCGAAGTTATCAGGAACACAGACTTCAGGGACATGACCTTTATCTCTGG840     GTATGCGAGGTTGCTATTTTCTAAAATCACCCCCTCCCTTATTTTTCACTTAAGGGACCT900     ATTTCTAAATTGTCTGAGGTCACCCCATCTTCAGATAATCTACCCTACATTCCTGGATCT960     TAAATACAAGGGCAGGAGGATTAGGATCCGTTTTTGAAGAAGCCAAAGTTGGAGGGTCGT1020     ATTTTGGCGTGCTACACCTACAGAATGAGTGAAATTAGAGGGCAGAAATAGGAGTCGGTA1080     GTTTTTTGTGGGTTGCCCTGTCCGGGCCCCTGGCATGCAGGCTTGGATGGAGGGAGAGGG1140     GTTGGGGGTTGCGGGGGACCGCGTTTGAAGTTGGGTCGGGCCAGCTGCTGTTCTCCTTAA1200     TAACGAGAGGGGAAAAGGAGGGAGGGAGGGAGAGATTGAAAGGAGGAGGGGAGGACCGGG1260     AGGGGAGGAAAGGGGAGGAGGAACCAGAGCGGGGAGCGCGGGGAGAGGGAGGAGAGCTAA1320     CTGCCCAGCCAGCTTCGGTCACGCTTCAGAGCGGAGAAGAGCGAGCAGGGGAGAGCGAGA1380     CCAGTTTTAAGGGGAGGACCGGTGCGAGTGAGGCAGCCCCTAGGCTCTGCTCGCCCACCA1440     CCCAATCCTCGCCTCCCTTCTGCTCCACCTTCTCTCTCTGCCCTCACCTCTCCCCCGAAA1500     ACCCCCTATTTAGCCAAAGGAAGGAGGTCAGGGAACGCTCTCCCCTCCCCTTCCAAAAAA1560     CAAAAACAGAAAAACCCTTTTCCAGGCCGGGGAAAGCAGGAGGGAGAGGGCGCGGGCTGC1620     CATG1624     (2) INFORMATION FOR SEQ ID NO:36:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 3158 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:     GAGCTCCCGTCCCCATACTACAGGTTCACATCCAGCTTTCAGGACTAGTCAGTCTATGTG60     GCCCTCCCTCAATTAATAAATCAGCAACTAATTTGCCAGGTGCGGTGGTTTGTGCCTGTA120     ATCCCAGCACTTTAGGAAGCTGAGGCAGGCAGATCACTTGAGGTCAGGAGTTCGAGACCA180     GCCTGGCCAACATGGTGAAATCCCGTATCTACTGAAAATACAAAAATTAGCCGGGCATGG240     TGGTATGCACCCGTAATCCCAGCTACTCAGGAAGCTGAGGCAGGAGAATCACTTGAAACC300     GGGAGGCAGAGGTTGCAGTAAGCTGCACTCCAGCCTGGTGACAAGAGCAAAACTTTGTGT360     CAAAAAAACAAAGAAAACCAAAAAACAAAGGAAAACACAAAAAACCCTTCTATTTGTTAA420     AAAAAAAAAAATCCACCGTGAACCAAAAATTAGTAAAAACAATGAACTAAAATTTTGTTT480     TTGCAAAATGTATGATAACAAAATGTTAAGGAAGGTCATGTGCCGTTATGGTTCACTGCA540     GCCTTGAACTCCTGGGCTCAAGCGATCCTCCTGCTTCGGTCTCCCTAGTAGCTGGGACTA600     CAGGCTTGTGCCACCGCACCCAGCTTATTTTTTTTTTTTATTTTTTGTAGAGATAGGAGT660     CTTGCTTTGTTGTCCAGGCTGGTCTTCAACTCCTAGCTTCCAGTGATCCTCCTGCCTCAG720     CCTCCCAAGTGCTGGGCCTGATGGGACATTTTTATACATAGTGCCATGTACCTATAAATG780     AGAAGTTTTAAAAATACTGATTTTAAAAATTAATTTATGTCAAGAATTTTTATACCAAAG840     TTAAAAAACCAAACCGAAAATATGAAAAGGGTTAATATCTTTGAGAGGTGATGAGAACTT900     ATAAGTCAATAAGAGAAAACAAACATCCCTATAAATGAATAAGCTAAGGACATGAATGGG960     TAATGTACATAAGAAATGTAAATGTCTAGTAATATGCCAAAATAGATTTATTATTACTAA1020     TAAGCCACTTTCACTCTCTAGTTGGCAGAGTTGTTTTGAAAAATAGATATGTAATGATGG1080     TGGAAAAGATTGGTTTAACTATTCAGCAGGAAAATTTGGCAATTAGAAGTGTATCAAAAG1140     CCTTAGAATGTTTCATAACCTTAGATTGGGAAATTCCACTTCTAGAAATTAATTCACTTC1200     TAGAAATAATCATGAGTGTGCACAAAGATATTACCACAAAAATATTTTACAGTATTATGT1260     CTAATAGAGAAGAACTAGAAATAATTTAAATTTCCACCAATACAGGTTTGCCAAAATACA1320     TTTTGTACATTCACCTAATGGTATATTATGTCCCTATTACAAATTACGTCCTAGAATATT1380     TAATAGCATGGAAAAGTGTTAACAGTATTTTTTTAATGAAAAAAGCTTACAAAACAGTTT1440     GTGATGATTCCATTTAAAATGTGTGTTTATTCATAGAACAAAGATTAGAAAAATAAACAT1500     TGATATATTAAAGGGTTATTTCATGGCAAATTGCAAATGATTATTTCCTTTTTTTGTGGC1560     TTATTTGTATTTTTGAAGTTTTCTACAATGTAAAAGAATATTTTATGATATGAAAACTAC1620     AATACAATTTATAATATAAGAAAGAATAATTCGGCCGGGAACGGTGGCTCACGCCTGTAA1680     TCCCAGCACTTTTGGAGGCCGAGACCGGCGGATCACGAGGTCAGGGGTTCAAGACTAGCC1740     TGGCCAACATAGTGAAACCCCATCTCTACGAAAAATACAAAAATTAGTCAGGCATGGTGG1800     TGCGTGCCTGTAGTCCCAGCTACTCGGGAATTGCTTGAACCCGGGAGGTGGAGGTTGCAG1860     TGAGCCCAGATCGCACCACTGCACTCCAGCTTGAGCAACAGAGTAGACTTCGTCTCAAAA1920     AAAAAAAAAAAAAAAAAAAGAATAATTAACAGAAAATGGTTAGACACTTCCTTAGTGTCT1980     CCTAAGTCAGGAGGACCCCAGTAGGGCAGGGATCCTCATGGCCTCCTCCCATTTGGAGCA2040     TTATTGGAGGTCTTTTTCGGCCTCTTCGTCAAGTGGAATCTAGCTTCCGGTAAAACTACA2100     AAGTAACCAAAAGTTTGGGAGGTGGAAGAAATGCAACCGGTAGATCTCACAGAGTCTGTG2160     CAAGAAACTGATTCAATGAGAATCTAGTTTCTCCGTCCACAGTTTCTCCAAACAGAAACT2220     AAGGCCGACTTTAGGGGCTTGTCCAAACCTAGGCAAGCAACTTAACAAGGTGAGGCCATG2280     ACTCCATGGCCTTTCCGTTCTGTTATATGCTGACTTAGACTAAAGCTCTCATACTTTAAA2340     GTGCACAGAAATCTAGTTAAAATGCAGATTCTGATTCAGGTTAGGGGTGGGCCTGAGAGT2400     CTGCATTTCTAACCAGCTCCCAGGCGATGACCACGCACGGGACAGGTCTGGGATCACAGT2460     TTAACTAGCAATGGTGTAGAACACAGAATCTGCAGCAAGAAGGCCAGCTTCCCAATCCTA2520     GCTCTGCCACGGACCAACTGAATGACAGTTGCCTCGGTTTCCGAGTTTTCGTGAAGATGT2580     AGTGAGTCATTACATCGTGAGGCTTTCGAGCAGCGTTCACTAAGAACTAGCTCTGACATT2640     ATTTATCGCATTCCTTAGAGCAAGCAGCCGGTGAAGTAGGGTTTGACGAATGAATAAGTG2700     AATGAATGACCTTTGGAGAAAAATTGTTTCCTGGGTGACTAGAGTCCGAGAAGCAAAATG2760     GGAGGGCCCGTGGTGGGTAGGAGGCCCACCTCCTAGAAAGTTCTCTGCACCCGGTGGTCC2820     AGAGGGCCTGGAGTGCCGGAAGCCGGCCGCGTTGCGCTCACGGCCCAATGGGGCCGCGGG2880     AGGGAGGGGAGAGCGCTCAGCCAACCCTTTCCGTTCCGGGCGCCGCAGCCCCGCCCCTCG2940     GAGCGTTGCGACGTCCGAGCATTCCACGGTTGCTACATCGTCGCGAGGGGGGGCGCCTGT3000     CAGGGAAGCGGCGCGCGCGCGGGCGGCGGGCGGGCTGGGGATCCGCCGCGCAGTGCCAGC3060     GCCAGCGCCAGACCCGCGCCCCGCGCTCTCCGGCCCGTCGCCTGTCTTGGGACTCGCGAG3120     CCCGCACTCCCGCCCTGCCTGTTCGCTGCCCGAGTATG3158     (2) INFORMATION FOR SEQ ID NO:37:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1089 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: double     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:     (A) NAME/KEY: CDS     (B) LOCATION: 13..888     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:     CCGGCCGCCGCGATGCAGAAATACGAGAAACTGGAAAAGATTGGGGAA48     MetGlnLysTyrGluLysLeuGluLysIleGlyGlu     1510     GGCACCTACGGAACTGTGTTCAAGGCCAAAAACCGGGAGACTCATGAG96     GlyThrTyrGlyThrValPheLysAlaLysAsnArgGluThrHisGlu     152025     ATCGTGGCTCTAAAACGGGTGAGGCTGGATGACGATGATGAGGGTGTG144     IleValAlaLeuLysArgValArgLeuAspAspAspAspGluGlyVal     303540     CCGAGTTCCGCCCTCCGGGAGATCTGCCTACTCAAGGAGCTGAAGCAC192     ProSerSerAlaLeuArgGluIleCysLeuLeuLysGluLeuLysHis     45505560     AAGAACATCGTCAGGCTTCATGACGTCCTGCACAGCGACAAGAAGCTG240     LysAsnIleValArgLeuHisAspValLeuHisSerAspLysLysLeu     657075     ACTTTGGTTTTTGAATTCTGTGACCAGGACCTGAAGAAGTATTTTGAC288     ThrLeuValPheGluPheCysAspGlnAspLeuLysLysTyrPheAsp     808590     AGTTGCAATGGTGACCTCGATCCTGAGATTGTAAAGTCATTCCTCTTC336     SerCysAsnGlyAspLeuAspProGluIleValLysSerPheLeuPhe     95100105     CAGCTACTAAAAGGGCTGGGATTCTGTCATAGCCGCAATGTGCTACAC384     GlnLeuLeuLysGlyLeuGlyPheCysHisSerArgAsnValLeuHis     110115120     AGGGACCTGAAGCCCCAGAACCTGCTAATAAACAGGAATGGGGAGCTG432     ArgAspLeuLysProGlnAsnLeuLeuIleAsnArgAsnGlyGluLeu     125130135140     AAATTGGCTGATTTTGGCCTGGCTCGAGCCTTTGGGATTCCCGTCCGC480     LysLeuAlaAspPheGlyLeuAlaArgAlaPheGlyIleProValArg     145150155     TGTTACTCAGCTGAGGTGGTCACACTGTGGTACCGCCCACCGGATGTC528     CysTyrSerAlaGluValValThrLeuTrpTyrArgProProAspVal     160165170     CTCTTTGGGGCCAAGCTGTACTCCACGTCCATCGACATGTGGTCAGCC576     LeuPheGlyAlaLysLeuTyrSerThrSerIleAspMetTrpSerAla     175180185     GGCTGCATCTTTGCAGAGCTGGCCAATGCTGGGCGGCCTCTTTTTCCC624     GlyCysIlePheAlaGluLeuAlaAsnAlaGlyArgProLeuPhePro     190195200     GGCAATGATGTCGATGACCAGTTGAAGAGGATCTTCCGACTGCTGGGG672     GlyAsnAspValAspAspGlnLeuLysArgIlePheArgLeuLeuGly     205210215220     ACGCCCACCGAGGAGCAGTGGCCCTCTATGACCAAGCTGCCAGACTAT720     ThrProThrGluGluGlnTrpProSerMetThrLysLeuProAspTyr     225230235     AAGCCCTATCCGATGTACCCGGCCACAACATCCCTGGTGAACGTCGTG768     LysProTyrProMetTyrProAlaThrThrSerLeuValAsnValVal     240245250     CCCAAACTCAATGCCACAGGGAGGGATCTGCTGCAGAACCTTCTGAAG816     ProLysLeuAsnAlaThrGlyArgAspLeuLeuGlnAsnLeuLeuLys     255260265     TGTAACCCTGTCCAGCGTATCTCAGCAGAAGAGGCCCTGCAGCACCCC864     CysAsnProValGlnArgIleSerAlaGluGluAlaLeuGlnHisPro     270275280     TACTTCTCCGACTTCTGTCCGCCCTAGGCCCGGGACCCCCGGCCTCAGCTGGGC918     TyrPheSerAspPheCysProPro     285290     CTGGCCTATTTAAGCCCCTCTTGAGAGGGGTGAGACAGTGGGGGTGCCTGGTGCGCTGTG978     CTCAGCAGTGCTGGGCCAGCCGGGGTGGGGTGCCTGAGCCCGAATTTCTCACTCCCTTTG1038     TGGACTTTATTTAATTTCATAAATTGGCTCCTTTCCCACAAAAAAAAAAGG1089     (2) INFORMATION FOR SEQ ID NO:38:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 292 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:     MetGlnLysTyrGluLysLeuGluLysIleGlyGluGlyThrTyrGly     151015     ThrValPheLysAlaLysAsnArgGluThrHisGluIleValAlaLeu     202530     LysArgValArgLeuAspAspAspAspGluGlyValProSerSerAla     354045     LeuArgGluIleCysLeuLeuLysGluLeuLysHisLysAsnIleVal     505560     ArgLeuHisAspValLeuHisSerAspLysLysLeuThrLeuValPhe     65707580     GluPheCysAspGlnAspLeuLysLysTyrPheAspSerCysAsnGly     859095     AspLeuAspProGluIleValLysSerPheLeuPheGlnLeuLeuLys     100105110     GlyLeuGlyPheCysHisSerArgAsnValLeuHisArgAspLeuLys     115120125     ProGlnAsnLeuLeuIleAsnArgAsnGlyGluLeuLysLeuAlaAsp     130135140     PheGlyLeuAlaArgAlaPheGlyIleProValArgCysTyrSerAla     145150155160     GluValValThrLeuTrpTyrArgProProAspValLeuPheGlyAla     165170175     LysLeuTyrSerThrSerIleAspMetTrpSerAlaGlyCysIlePhe     180185190     AlaGluLeuAlaAsnAlaGlyArgProLeuPheProGlyAsnAspVal     195200205     AspAspGlnLeuLysArgIlePheArgLeuLeuGlyThrProThrGlu     210215220     GluGlnTrpProSerMetThrLysLeuProAspTyrLysProTyrPro     225230235240     MetTyrProAlaThrThrSerLeuValAsnValValProLysLeuAsn     245250255     AlaThrGlyArgAspLeuLeuGlnAsnLeuLeuLysCysAsnProVal     260265270     GlnArgIleSerAlaGluGluAlaLeuGlnHisProTyrPheSerAsp     275280285     PheCysProPro     290     (2) INFORMATION FOR SEQ ID NO:39:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:     TGGATGYTNGRAGTNTGYGAMGARCARAARTGYGARGA38     (2) INFORMATION FOR SEQ ID NO:40:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 13 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:     TrpMetLeuGluValCysGluGluGlnLysCysGluGlu     1510     (2) INFORMATION FOR SEQ ID NO:41:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 28 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:     GlyThrAsnThrThrTyrCysCysAsnTyrThrAsnGlyCysAsnAla     151015     ThrGlyAlaAlaTyrThrAlaTyrThrAsnGlyAla     2025     (2) INFORMATION FOR SEQ ID NO:42:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 10 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:     ValPheProLeuAlaMetAsnTyrLeuAsp     1510     (2) INFORMATION FOR SEQ ID NO:43:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:     RTCNGTRTADATRCANARYTTYTC24     (2) INFORMATION FOR SEQ ID NO:44:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 8 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:     GluLysLeuCysIleTyrThrAsp     15     (2) INFORMATION FOR SEQ ID NO:45:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 9 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:     CysTyrPheSerAspPheCysProPro     15     (2) INFORMATION FOR SEQ ID NO:46:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 amino acids     (B) TYPE: amino acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:     CysAspGluLeuAspGlnAlaSerThrProThrAspValArgAspIle     151015     AspLeu     (2) INFORMATION FOR SEQ ID NO:47:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 122 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:     CysSerTyrPheLysCysValGlnLysAlaIleGlnProTyrMetHis     151015     ArgMetValProLeuLeuMetValMetLeuGluValCysGluGluGln     202530     LysCysGluGluLysValPheProLeuAlaThrIleTyrLeuAspCys     354045     PhePheAlaArgIleProThrSerLysSerHisLeuGlnLeuLeuGly     505560     AlaValCysMetPheLeuAlaSerArgLeuLysGluSerSerProLeu     65707580     ThrAlaLysLysLeuCysIleTyrThrAspAsnSerIleLysProGln     859095     GluLeuLeuGluTrpGluLeuValValLeuGlyLysLeuLysTrpAsn     100105110     LeuAlaAlaValThrProHisAspPheIle     115120     (2) INFORMATION FOR SEQ ID NO:48:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 104 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: internal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:     TyrIleLeuHisLysLeuProGlnGlnArgGluLysLeuSerAlaMet     151015     TyrProProSerMetValAlaThrGlySerValGlyAlaAlaIleCys     202530     GlyLeuGlnGlnHisGluGluValSerSerLeuProCysAsnAlaLeu     354045     ThrGluLeuLeuAlaLysIleThrAsnThrAspValAspCysLeuLys     505560     AlaAsnArgGluHisIleGluValValPheLeuAsnSerLeuGlnGln     65707580     CysHisGlnAspGlnGlnAspArgSerLysSerGluAspGluLeuGly     859095     GlnAlaSerThrProIleAspLeu     100     (2) INFORMATION FOR SEQ ID NO:49:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 4 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: C-terminal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:     AspIleAspLeu     (2) INFORMATION FOR SEQ ID NO:50:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 174 amino acids     (B) TYPE: amino acid     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (v) FRAGMENT TYPE: C-terminal     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:     ArgCysValHisCysAlaTyrPheGlnCysValGlnArgGluSerLys     151015     ProHisMetArgLysMetLeuValTyrTrpMetLeuGluValCysGlu     202530     GluGlnCysCysGluGluGluGlnCysCysLysGluGluValPhePro     354045     LeuAlaMetAsnHisLeuHisAlaThrCysProThrSerProProThr     505560     ArgLysAlaGlnLeuGlnLeuLeuValAlaValSerMetArgLeuAla     65707580     SerLysLeuArgLysThrGlyProMetThrIleGluLysMetCysIle     859095     TyrThrAspHisAlaValSerProCysGlnLeuArgAspTrpGluVal     100105110     MetValLeuGlyLysLeuLysTrpAspLeuAlaAlaValIleAlaHis     115120125     AspPheLeuAlaLeuIleLeuHisArgArgGlnAlaLeuValLysLys     130135140     HisAlaGlnIlePheLeuAlaValCysAlaThrAspTyrThrPheAla     145150155160     MetTyrProProSerSerCysGluAsnAsnProAsnAlaCys     165170     __________________________________________________________________________ 

I claim:
 1. Isolated nucleic acid encoding a D2-type cyclin of mammalian origin of approximate molecular weight 33-34 kD, which nucleic acid comprises a nucleotide sequence represented by SEQ ID NO.
 4. 2. Isolated DNA encoding a mammalian D2-type cyclin which is active in G1 phase and which replaces a CLN gene essential for cell cycle START in budding yeast, which DNA selectively hybridizes under high stringency conditions to the nucleic acid represented by SEQ ID NO:
 3. 3. The nucleic acid of claim 2, which is nucleic acid is labelled.
 4. The nucleic acid of claim 3, wherein the label is selected from the group consisting of: radioactive labels, fluorescent labels, enzymatic labels and binding pair members.
 5. An isolated nucleic acid comprising a nucleotide sequence encoding a D2-type cyclin represented by SEQ ID No.
 3. 6. An isolated nucleic acid comprising a nucleotide sequence represented in SEQ ID No: 3, or a complementary sequence thereof.
 7. The nucleic acid of any of claims 1, 2 or 6, which nucleic acid encodes a D2-type cyclin able to bind to a cyclin dependent kinase (CDK).
 8. The nucleic acid of claim 7, wherein the CDK is CDK4.
 9. The nucleic acid of claim 6, which nucleic acid is labelled.
 10. The nucleic acid of claim 9, wherein the label is selected from the group consisting of: radioactive labels, fluorescent labels, enzymatic labels and binding pair members.
 11. An isolated nucleic acid comprising a cyclin D2 nucleotide sequence represented in SEQ ID No: 3, or a complementary sequence thereof, which cyclin D2 nucleotide sequence specifically hybridizes to a D2 type cyclin gene and inhibits expression of said gene.
 12. Isolated nucleic acid comprising a D2-type cyclin coding region of the nucleic acid sequence represented by SEQ ID No. 3, which nucleic acid encodes a D-type cyclin able to bind to a cyclin dependent kinase (CDK).
 13. Isolated nucleic acid encoding a D3-type cyclin of mammalian origin of approximate molecular weight 33-34 kD, which nucleic acid comprises a nucleotide sequence represented by SEQ ID NO.
 6. 14. Isolated DNA encoding a mammalian D3-type cyclin which is active in G1 phase and which replaces a CLN gene essential for cell cycle START in budding yeast, which DNA selectively hybridizes under high stringency conditions to the nucleic acid represented by SEQ ID NO:
 5. 15. The nucleic acid of claim 14, which is nucleic acid is labelled.
 16. The nucleic acid of claim 15, wherein the label is selected from the group consisting of: radioactive labels, fluorescent labels, enzymatic labels and binding pair members.
 17. An isolated nucleic acid comprising a nucleotide sequence encoding a D3-type cyclin represented by SEQ ID No.
 5. 18. An isolated nucleic acid comprising a nucleotide sequence represented in SEQ ID No: 5, or a complementary sequence thereof.
 19. The nucleic acid of any claims 13, 14 or 18, which nucleic acid encodes a D3-type cyclin able to bind to a cyclin dependence kinase (CDK).
 20. The nucleic acid of claim 19, wherein the CDK is CDK4.
 21. The nucleic acid of claim 18, which nucleic acid is labelled.
 22. The nucleic acid of claim 21, wherein the label is selected from the group consisting of: radioactive labels, fluorescent labels, enzymatic labels and binding pair members.
 23. An isolated nucleic acid comprising a cyclin D3 nucleotide sequence represented in SEQ ID No: 5, or a complementary sequence thereof, which cyclin D3 nucleotide sequence specifically hybridizes to a D3 type cyclin gene and inhibits expression of said gene.
 24. Isolated nucleic acid comprising a D3-type cyclin coding region of the nucleic acid sequence represented by SEQ ID No. 5, which nucleic acid encodes a D-type cyclin able to bind to a cyclin dependent kinase (CDK).
 25. Isolated nucleic acid encoding a D2-type cyclin of human origin of approximate molecular weight 33-34 kD.
 26. Isolated nucleic acid encoding a D3-type cyclin of human origin of approximate molecular weight 33-34 kD.
 27. Isolated DNA encoding a D2-type cyclin which binds to bind to a cyclin dependent kinase (CDK), which DNA hybridizes under high stringency conditions to the nucleic acid represented by SEQ ID NO:
 3. 28. Isolated DNA encoding a D3-type cyclin which binds to bind to a cyclin dependent kinase (CDK), which DNA hybridizes under high stringency conditions to the nucleic acid represented by SEQ ID NO:
 5. 29. An expression vector for recombinantly producing the D2-type cyclin, comprising the nucleic acid of any of claims 1, 2, 5, 12 or
 27. 30. An expression vector for recombinantly producing the D3-type cyclin, comprising the nucleic acid of any of claims 13, 14, 17, 24 or
 28. 31. The nucleic acid of claim 12 or 27, wherein the CDK is CDK4.
 32. A recombinant cell transformed with the expression vector of claim
 29. 33. The nucleic acid of claim 24 or 28, wherein the CDK is CDK4.
 34. A recombinant cell transformed with the expression vector of claim
 30. 35. The cell of claim 32 wherein the cell is a eukaryotic cell.
 36. The nucleic acid of any of claims 1, 2, 6, 12 or 27 which D2-type cyclin binds to and regulates a kinase activity of said CDK.
 37. The nucleic acid of any of claims 13, 14, 18, 24 or 28 which D3-type cyclin binds to and regulates a kinase activity of said CDK.
 38. The cell of claim 34 wherein the cell is a eukaryotic cell. 